Compositions and uses for treatment thereof

ABSTRACT

The invention is directed generally to oligonucleotide compositions for the treatment of DNA repeat expansion diseases. The invention also relates to oligonucleotides directed to subunits of the DNA mismatch repair system.

This application is a continuation-in-part of PCT Application No.PCT/US2015/029724, filed May 7, 2015, which claims priority to U.S.Provisional Patent Application Ser. No. 61/989,898, filed May 7, 2014,and the entire disclosures of which are incorporated by reference intheir entireties.

All patents, patent applications and publications cited herein arehereby incorporated by reference in their entirety. The disclosures ofthese publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art as known to those skilled therein as of the date of theinvention described and claimed herein

This patent disclosure contains material that is subject to copyrightprotection. The copyright owner has no objection to the facsimilereproduction by anyone of the patent document or the patent disclosureas it appears in the U.S. Patent and Trademark Office patent file orrecords, but otherwise reserves any and all copyright rights.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 9, 2015, isnamed 2932719.3.W01_SL.txt and is 949,305 bytes in size.

FIELD OF THE INVENTION

The present invention relates generally to compositions for thetreatment of DNA repeat expansion diseases. In one embodiment, thepresent disclosure relates to oligonucleotides directed to subunits ofthe DNA mismatch repair system. In one embodiment, the presentdisclosure relates to oligonucleotides directed at skipping of MLH3 exon7 to slow the progression of repeat expansion disorders.

BACKGROUND OF THE INVENTION

Genomic instability underlies an increasing number of disorders, plays amajor role in cancer and contributes to aging. DNA mismatch repair (MMR)is essential for maintaining genome integrity. However, when it comes tocertain types of repetitive DNA, MMR actually contributes to genomeinstability. MMR has been implicated in repeat expansions of numerousdisorders including Huntington's disease (HD) and myotonic dystrophy(DM). Friedreich ataxia (FRDA), the most common inherited ataxia, is aprogressive neurodegenerative disorder caused by GAA⋅TTC repeatexpansion in the first intron of the frataxin (FXN) gene. Currentlythere is no treatment and no cure for Friedreich ataxia or any of themany other DNA repeat expansion diseases. While each of the individualrepeat expansion diseases is rare or not necessarily common, inaggregate, the victims of the currently known repeat expansion diseasesnumber over 100,000 in the United States alone.

SUMMARY OF THE INVENTION

The invention is directed to therapeutics useful to slow the expansionrate in repeat expansion diseases. In one embodiment, a centralmechanism is likely shared by all repeat expansion diseases thus usefulin the treatment of many, if not all of the diseases in this class. Forprogressive repeat expansion diseases such as Friedreich ataxia orHuntington's disease MLH3 exon skipping may make it possible to delay oreven prevent the onset of symptoms if treatment is started early.Chemically similar morpholino splice switching oligonucleotides (SSOs)are currently in human trials for exon skipping in Duchenne musculardystrophy.

An aspect of the invention is directed to an isolated nuclease-resistantoligonucleotide comprising a nucleic acid sequence that hybridizes to acomplementary target nucleic acid sequence of a gene or gene productencoding a component of a mismatch repair (MMR) complex. For example,the oligonucleotide comprises a sequence that specifically hybridizes ina human cell with a nucleic acid sequence encoding a subunit of the MMRsystem (e.g., MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2). In oneembodiment, the nucleic acid sequence is a complementary sequence for ahuman MLH3 gene or gene product. In one embodiment, thenuclease-resistant oligonucleotide is useful for inducing exon skipping.For example, oligonucleotide(s) can induce skipping of MSH2 exon 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or a combination ofMSH2 exons. Oligonucleotide(s) can induce skipping of MSH3 exon 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or a combination of MSH3 exons. Oligonucleotide(s) can induceskipping of MSH6 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or a combination ofMSH6 exons. Oligonucleotide(s) can induce skipping of MLH1 exon 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or acombination of MLH1 exons. Oligonucleotide(s) can induce skipping ofMLH3 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or a combination ofMLH3 exons. Oligonucleotide(s) can induce skipping of PMS1 exon 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or a combination of PMS1 exons.Oligonucleotide(s) can induce skipping of PMS2 exon 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, or a combination of PMS2 exons. In oneembodiment, the oligonucleotide directs skipping of one or more exons ofMSH2, MSH3, MSH6, PMS1, PMS2, MLH1, or MLH3. In one embodiment, one ormore oligonucleotides can induce skipping of MLH3 exon 7. In oneembodiment, the oligonucleotide is decreases the rate of DNA repeatexpansion. In one embodiment, the oligonucleotide is useful in treatinga DNA repeat expansion disease. In one embodiment, the target nucleicacid sequence is located on human chromosome 2, 3, 5, 7, or 14. In oneembodiment, the target nucleic acid sequence (or target complementarynucleic acid sequence) comprises: a nucleic acid sequence correspondingto a region of interest for any one of the exons 1-16 described hereinfor GenBank Accession No. NG 007110.2, or to an intron-exon junction, orto an exon-intron junction listed with GenBank Accession No.NG_007110.2; a nucleic acid sequence corresponding to a region ofinterest for any one of the exons 1-24 described herein for GenBankAccession No. NG_016607.1, or to an intron-exon junction, or to anexon-intron junction listed with GenBank Accession No. NG_016607.1; anucleic acid sequence corresponding to a region of interest for any oneof the exons 1-10 described herein for GenBank Accession No. NG_007111.1or SEQ ID NO: 33, or to an intron-exon junction, or to an exon-intronjunction listed with GenBank Accession No. NG_007111.1 or SEQ ID NO: 33;a nucleic acid sequence corresponding to a region of interest for anyone of the exons 1-19 described herein for GenBank Accession No.NG_007109.2, or to an intron-exon junction, or to an exon-intronjunction listed with GenBank Accession No. NG_007109.2; a nucleic acidsequence corresponding to a region of interest for any one of the exons1-13 described herein for GenBank Accession No. NG_008648.1, or to anintron-exon junction, or to an exon-intron junction listed with GenBankAccession No. NG_008648.1; a nucleic acid sequence corresponding to aregion of interest for any one of the exons 1-13 described herein forGenBank Accession No. NG_008649.1 or SEQ ID NO: 1, or to an intron-exonjunction, or to an exon-intron junction listed with GenBank AccessionNo. NG_008649.1 or SEQ ID NO: 1; or a nucleic acid sequencecorresponding to a region of interest for any one of the exons 1-15described herein for GenBank Accession No. NG_008466.1, or to anintron-exon junction, or to an exon-intron junction listed with GenBankAccession No. NG_008466.1. In one embodiment, the component of the MMRcomplex comprises MutS or MutL. In one embodiment, MutS comprises asubunit selected from the group consisting of MSH2, MSH3, and MSH6. Inone embodiment, MutL comprises a subunit selected from the groupconsisting of MLH1, MLH3, PMS1, and PMS2. In one embodiment, MLH3comprises SEQ ID NO: 1. In one embodiment, the oligonucleotidehybridizes to the target complementary nucleic acid sequence comprisingSEQ ID NO: 2. In one embodiment, the oligonucleotide has at least 60%,at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% identity toa nucleic acid sequence comprising SEQ ID NO: 3 or SEQ ID NO: 4. In oneembodiment, the oligonucleotide has at least 60%, at least 65%, at least70%, at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% identity to a nucleic acidsequence depicted in Table 4. In one embodiment, the oligonucleotidecomprises 15 to 30 nucleotide bases in length. In one embodiment, theoligonucleotide comprises one or more morpholino subunits, one or morelocked nucleic acid subunits, one or more 2-O-methyl moieties, or one ormore peptide moieties.

An aspect of the invention is directed to a pharmaceutical compositioncomprising a nuclease-resistant oligonucleotide comprising a nucleicacid sequence that hybridizes to a complementary target nucleic acidsequence of a gene or gene product encoding a component of a mismatchrepair (MMR) complex, and a pharmaceutically acceptable carrier. Forexample, the oligonucleotide comprises a sequence that specificallyhybridizes in a human cell with a nucleic acid sequence encoding asubunit of the MMR system (e.g., MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, orPMS2). In one embodiment, the nucleic acid sequence is a complementarysequence for a human MLH3 gene or gene product. In one embodiment, thenuclease-resistant oligonucleotide is useful for inducing exon skipping.For example, oligonucleotide(s) can induce skipping of MSH2 exon 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or a combination ofMSH2 exons. Oligonucleotide(s) can induce skipping of MSH3 exon 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or a combination of MSH3 exons. Oligonucleotide(s) can induceskipping of MSH6 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or a combination ofMSH6 exons. Oligonucleotide(s) can induce skipping of MLH1 exon 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or acombination of MLH1 exons. Oligonucleotide(s) can induce skipping ofMLH3 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or a combination ofMLH3 exons. Oligonucleotide(s) can induce skipping of PMS1 exon 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or a combination of PMS1 exons.Oligonucleotide(s) can induce skipping of PMS2 exon 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, or a combination of PMS2 exons. In oneembodiment, the oligonucleotide directs skipping of one or more exons ofMSH2, MSH3, MSH6, PMS1, PMS2, MLH1, or MLH3. In one embodiment, one ormore oligonucleotides can induce skipping of MLH3 exon 7. In oneembodiment, the oligonucleotide decreases the rate of DNA repeatexpansion. In one embodiment, the oligonucleotide is useful in treatinga DNA repeat expansion disease. In one embodiment, the target nucleicacid sequence is located on human chromosome 2, 3, 5, 7, or 14. In oneembodiment, the target nucleic acid sequence (or target complementarynucleic acid sequence) comprises: a nucleic acid sequence correspondingto a region of interest for any one of the exons 1-16 described hereinfor GenBank Accession No. NG_007110.2, or to an intron-exon junction, orto an exon-intron junction listed with GenBank Accession No.NG_007110.2; a nucleic acid sequence corresponding to a region ofinterest for any one of the exons 1-24 described herein for GenBankAccession No. NG_016607.1, or to an intron-exon junction, or to anexon-intron junction listed with GenBank Accession No. NG_016607.1; anucleic acid sequence corresponding to a region of interest for any oneof the exons 1-10 described herein for GenBank Accession No. NG_007111.1or SEQ ID NO: 33, or to an intron-exon junction, or to an exon-intronjunction listed with GenBank Accession No. NG_007111.1 or SEQ ID NO: 33;a nucleic acid sequence corresponding to a region of interest for anyone of the exons 1-19 described herein for GenBank Accession No.NG_007109.2, or to an intron-exon junction, or to an exon-intronjunction listed with GenBank Accession No. NG_007109.2; a nucleic acidsequence corresponding to a region of interest for any one of the exons1-13 described herein for GenBank Accession No. NG_008648.1, or to anintron-exon junction, or to an exon-intron junction listed with GenBankAccession No. NG_008648.1; a nucleic acid sequence corresponding to aregion of interest for any one of the exons 1-13 described herein forGenBank Accession No. NG_008649.1 or SEQ ID NO: 1, or to an intron-exonjunction, or to an exon-intron junction listed with GenBank AccessionNo. NG_008649.1 or SEQ ID NO: 1; or a nucleic acid sequencecorresponding to a region of interest for any one of the exons 1-15described herein for GenBank Accession No. NG_008466.1, or to anintron-exon junction, or to an exon-intron junction listed with GenBankAccession No. NG_008466.1. In one embodiment, the component of the MMRcomplex comprises MutS or MutL. In one embodiment, MutS comprises asubunit selected from the group consisting of MSH2, MSH3, and MSH6. Inone embodiment, MutL comprises a subunit selected from the groupconsisting of MLH1, MLH3, PMS1, and PMS2. In one embodiment, MLH3comprises SEQ ID NO: 1. In one embodiment, the oligonucleotidehybridizes to the target complementary nucleic acid sequence comprisingSEQ ID NO: 2. In one embodiment, the oligonucleotide has at least 60%,at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% identity toa nucleic acid sequence comprising SEQ ID NO: 3 or SEQ ID NO: 4. In oneembodiment, the oligonucleotide has at least 60%, at least 65%, at least70%, at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% identity to a nucleic acidsequence depicted in Table 4. In one embodiment, the oligonucleotidecomprises 15 to 30 nucleotide bases in length. In one embodiment, theoligonucleotide comprises one or more morpholino subunits, one or morelocked nucleic acid subunits, one or more 2-O-methyl moieties, or one ormore peptide moieties.

An aspect of the invention is directed to a pharmaceutical compositioncomprising a nuclease-resistant oligonucleotide 15 to 30 nucleotidebases in length targeted to a complementary nucleic acid sequence of agene or gene product encoding a MutS or MutL subunit, wherein theoligonucleotide hybridizes with and decreases the expression of thehuman MutS or MutL subunit by at least 20%, and wherein theoligonucleotide comprises at least one modification. In one embodiment,the modification comprises a phosphorothioate backbone. In oneembodiment, the modification comprises a phosphorodiamidate morpholinonucleotide. In one embodiment, the modification results in acharge-negative oligonucleotide. In one embodiment, the modificationresults in a charge-neutral oligonucleotide. In one embodiment, themodification comprises a phosphorodiamidate morpholino nucleotide, or a2-aminoethylglycine functionalized nucleotide. In one embodiment, themodification comprises a phosphorothioate backbone, a 5-methylcytosinenucleotide, a 2′-O-methoxyethyl sugar moiety, a locked nucleic acidsubunit, an ethylene-bridged nucleic acid subunit, or a combinationthereof. In one embodiment, MutS comprises a subunit selected from thegroup consisting of MSH2, MSH3, and MSH6. In one embodiment, MutLcomprises a subunit selected from the group consisting of MLH1, MLH3,PMS1, and PMS2. In one embodiment, MLH3 comprises SEQ ID NO: 1. In oneembodiment, the oligonucleotide of the composition hybridizes to thetarget complementary nucleic acid sequence comprising SEQ ID NO: 2. Inone embodiment, the oligonucleotide has at least 60%, at least 65%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identity to a nucleicacid sequence comprising SEQ ID NO: 3 or SEQ ID NO: 4. In oneembodiment, the oligonucleotide has at least 60%, at least 65%, at least70%, at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% identity to a nucleic acidsequence depicted in Table 4. In one embodiment, the nuclease-resistantoligonucleotide of the composition is useful for inducing exon skipping.For example, oligonucleotide(s) can induce skipping of MSH2 exon 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or a combination ofMSH2 exons. Oligonucleotide(s) can induce skipping of MSH3 exon 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or a combination of MSH3 exons. Oligonucleotide(s) can induceskipping of MSH6 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or a combination ofMSH6 exons. Oligonucleotide(s) can induce skipping of MLH1 exon 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or acombination of MLH1 exons. Oligonucleotide(s) can induce skipping ofMLH3 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or a combination ofMLH3 exons. Oligonucleotide(s) can induce skipping of PMS1 exon 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or a combination of PMS1 exons.Oligonucleotide(s) can induce skipping of PMS2 exon 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, or a combination of PMS2 exons. In oneembodiment, the oligonucleotide of the composition directs skipping ofone or more exons of MSH2, MSH3, MSH6, PMS1, PMS2, MLH1, or MLH3. In oneembodiment, the oligonucleotide(s) of the composition can induceskipping of MLH3 exon 7. In one embodiment, the oligonucleotide of thecomposition hybridizes with and decreases the expression of the humanMutS or MutL subunit by at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%.

An aspect of the invention provides for an oligonucleotide complex formodulating the expression or activity of a gene or gene product encodinga component of a mismatch repair (MMR) system, the complex comprising afirst oligonucleotide and a second oligonucleotide, wherein the firstoligonucleotide comprises a sequence complementary to an acceptor regionof an exon of a gene encoding a MutS or MutL subunit, and wherein thenucleic acid sequence of the first oligonucleotide comprises anuclease-resistant modification, and wherein the second oligonucleotidecomprises a sequence complementary to a donor region of an exon of agene encoding a MutS or MutL subunit, and wherein the nucleic acidsequence of the second oligonucleotide comprises a nuclease-resistantmodification. An aspect of the invention is directed to anoligonucleotide complex for modulating the expression or activity of agene or gene product encoding a component of a mismatch repair (MMR)system, the complex comprising a first oligonucleotide and a secondoligonucleotide, wherein the first oligonucleotide comprises a sequencecomplementary to an acceptor region of an exon of a gene encoding a MutSor MutL subunit, and wherein the nucleic acid sequence of the firstoligonucleotide comprises a nuclease-resistant modification, and whereinthe second oligonucleotide comprises a sequence complementary to a donorregion of an exon of a gene encoding a MutS or MutL subunit. An aspectof the invention provides for an oligonucleotide complex for modulatingthe expression or activity of a gene or gene product encoding acomponent of a mismatch repair (MMR) system, the complex comprising afirst oligonucleotide and a second oligonucleotide, wherein the firstoligonucleotide comprises a sequence complementary to an acceptor regionof an exon of a gene encoding a MutS or MutL subunit, and wherein thesecond oligonucleotide comprises a sequence complementary to a donorregion of an exon of a gene encoding a MutS or MutL subunit, and whereinthe nucleic acid sequence of the second oligonucleotide comprises anuclease-resistant modification. In one embodiment, thenuclease-resistant modification comprises one or more morpholinosubunits, one or more locked nucleic acid subunits, one or more2-O-methyl moieties, one or more peptide moieties, or a combinationthereof. In one embodiment, the first oligonucleotide comprises anucleic acid sequence having at least 60%, at least 65%, at least 70%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identity to a nucleic acid sequencecomprising SEQ ID NO: 3 or SEQ ID NO: 4. In one embodiment, the firstoligonucleotide comprises a nucleic acid sequence having at least 60%,at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% identity toa nucleic acid sequence depicted in Table 4. In one embodiment, thefirst oligonucleotide is directed to a target nucleic acid sequence (ortarget complementary nucleic acid sequence) comprising: a nucleic acidsequence corresponding to a region of interest for any one of the exons1-16 described herein for GenBank Accession No. NG_007110.2, or to anintron-exon junction, or to an exon-intron junction listed with GenBankAccession No. NG_007110.2; a nucleic acid sequence corresponding to aregion of interest for any one of the exons 1-24 described herein forGenBank Accession No. NG_016607.1, or to an intron-exon junction, or toan exon-intron junction listed with GenBank Accession No. NG_016607.1; anucleic acid sequence corresponding to a region of interest for any oneof the exons 1-10 described herein for GenBank Accession No. NG_007111.1or SEQ ID NO: 33, or to an intron-exon junction, or to an exon-intronjunction listed with GenBank Accession No. NG_007111.1 or SEQ ID NO: 33;a nucleic acid sequence corresponding to a region of interest for anyone of the exons 1-19 described herein for GenBank Accession No.NG_007109.2, or to an intron-exon junction, or to an exon-intronjunction listed with GenBank Accession No. NG_007109.2; a nucleic acidsequence corresponding to a region of interest for any one of the exons1-13 described herein for GenBank Accession No. NG_008648.1, or to anintron-exon junction, or to an exon-intron junction listed with GenBankAccession No. NG_008648.1; a nucleic acid sequence corresponding to aregion of interest for any one of the exons 1-13 described herein forGenBank Accession No. NG_008649.1 or SEQ ID NO: 1, or to an intron-exonjunction, or to an exon-intron junction listed with GenBank AccessionNo. NG_008649.1 or SEQ ID NO: 1; or a nucleic acid sequencecorresponding to a region of interest for any one of the exons 1-15described herein for GenBank Accession No. NG_008466.1, or to anintron-exon junction, or to an exon-intron junction listed with GenBankAccession No. NG_008466.1. In one embodiment, the second oligonucleotidecomprises a nucleic acid sequence having at least 60%, at least 65%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identity to a nucleicacid sequence comprising SEQ ID NO: 3 or SEQ ID NO: 4. In oneembodiment, the second oligonucleotide comprises a nucleic acid sequencehaving at least 60%, at least 65%, at least 70%, at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% identity to a nucleic acid sequence depicted in Table 4. Inone embodiment, the second oligonucleotide is directed to a targetnucleic acid sequence (or target complementary nucleic acid sequence)comprising: a nucleic acid sequence corresponding to a region ofinterest for any one of the exons 1-16 described herein for GenBankAccession No. NG_007110.2, or to an intron-exon junction, or to anexon-intron junction listed with GenBank Accession No. NG_007110.2; anucleic acid sequence corresponding to a region of interest for any oneof the exons 1-24 described herein for GenBank Accession No.NG_016607.1, or to an intron-exon junction, or to an exon-intronjunction listed with GenBank Accession No. NG_016607.1; a nucleic acidsequence corresponding to a region of interest for any one of the exons1-10 described herein for GenBank Accession No. NG_007111.1 or SEQ IDNO: 33, or to an intron-exon junction, or to an exon-intron junctionlisted with GenBank Accession No. NG_007111.1 or SEQ ID NO: 33; anucleic acid sequence corresponding to a region of interest for any oneof the exons 1-19 described herein for GenBank Accession No.NG_007109.2, or to an intron-exon junction, or to an exon-intronjunction listed with GenBank Accession No. NG_007109.2; a nucleic acidsequence corresponding to a region of interest for any one of the exons1-13 described herein for GenBank Accession No. NG_008648.1, or to anintron-exon junction, or to an exon-intron junction listed with GenBankAccession No. NG_008648.1; a nucleic acid sequence corresponding to aregion of interest for any one of the exons 1-13 described herein forGenBank Accession No. NG_008649.1 or SEQ ID NO: 1, or to an intron-exonjunction, or to an exon-intron junction listed with GenBank AccessionNo. NG_008649.1 or SEQ ID NO: 1; or a nucleic acid sequencecorresponding to a region of interest for any one of the exons 1-15described herein for GenBank Accession No. NG_008466.1, or to anintron-exon junction, or to an exon-intron junction listed with GenBankAccession No. NG_008466.1. In one embodiment, MutS comprises a subunitselected from the group consisting of MSH2, MSH3, and MSH6. In oneembodiment, MutL comprises a subunit selected from the group consistingof MLH1, MLH3, PMS1, and PMS2. In one embodiment, MLH3 comprises SEQ IDNO: 1. In one embodiment, modulation of expression or activity is adecrease in the expression or activity of the human MutS or MutL subunitby at least 25%, at least 30%, at least 35%, at least 40%, at least 45%,at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%.

An aspect of the invention provides for a kit for the treatment of a DNARepeat Expansion Disease (DRED). In one embodiment, the kit comprises anoligonucleotide complex described herein and instructions for use. Inone embodiment, the oligonucleotides of the complex comprise a nucleicacid sequence having at least 60%, at least 65%, at least 70%, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% identity to a nucleic acid sequencecomprising SEQ ID NO: 3 or SEQ ID NO: 4. In one embodiment, theoligonucleotides if the complex comprise a nucleic acid sequence havingat least 60%, at least 65%, at least 70%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identity to a nucleic acid sequence depicted in Table 4. In oneembodiment, the oligonucleotides of the complex can be directed to atarget nucleic acid sequence (or target complementary nucleic acidsequence) comprising: a nucleic acid sequence corresponding to a regionof interest for any one of the exons 1-16 described herein for GenBankAccession No. NG_007110.2, or to an intron-exon junction, or to anexon-intron junction listed with GenBank Accession No. NG_007110.2; anucleic acid sequence corresponding to a region of interest for any oneof the exons 1-24 described herein for GenBank Accession No.NG_016607.1, or to an intron-exon junction, or to an exon-intronjunction listed with GenBank Accession No. NG_016607.1; a nucleic acidsequence corresponding to a region of interest for any one of the exons1-10 described herein for GenBank Accession No. NG_007111.1 or SEQ IDNO: 33, or to an intron-exon junction, or to an exon-intron junctionlisted with GenBank Accession No. NG_007111.1 or SEQ ID NO: 33; anucleic acid sequence corresponding to a region of interest for any oneof the exons 1-19 described herein for GenBank Accession No.NG_007109.2, or to an intron-exon junction, or to an exon-intronjunction listed with GenBank Accession No. NG_007109.2; a nucleic acidsequence corresponding to a region of interest for any one of the exons1-13 described herein for GenBank Accession No. NG_008648.1, or to anintron-exon junction, or to an exon-intron junction listed with GenBankAccession No. NG_008648.1; a nucleic acid sequence corresponding to aregion of interest for any one of the exons 1-13 described herein forGenBank Accession No. NG_008649.1 or SEQ ID NO: 1, or to an intron-exonjunction, or to an exon-intron junction listed with GenBank AccessionNo. NG_008649.1 or SEQ ID NO: 1; or a nucleic acid sequencecorresponding to a region of interest for any one of the exons 1-15described herein for GenBank Accession No. NG_008466.1, or to anintron-exon junction, or to an exon-intron junction listed with GenBankAccession No. NG_008466.1.

An aspect of the invention provides for a kit for the treatment of a DNARepeat Expansion Disease (DRED). In one embodiment, the kit comprises anuclease-resistant oligonucleotide compound as described herein andinstructions for use. In one embodiment, the oligonucleotide comprises anucleic acid sequence having at least 60%, at least 65%, at least 70%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identity to a nucleic acid sequencecomprising SEQ ID NO: 3 or SEQ ID NO: 4. In one embodiment, theoligonucleotide comprises a nucleic acid sequence having at least 60%,at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% identity toa nucleic acid sequence depicted in Table 4. In one embodiment, theoligonucleotide can be directed to a target nucleic acid sequence (ortarget complementary nucleic acid sequence) comprising: a nucleic acidsequence corresponding to a region of interest for any one of the exons1-16 described herein for GenBank Accession No. NG_007110.2, or to anintron-exon junction, or to an exon-intron junction listed with GenBankAccession No. NG_007110.2; a nucleic acid sequence corresponding to aregion of interest for any one of the exons 1-24 described herein forGenBank Accession No. NG_016607.1, or to an intron-exon junction, or toan exon-intron junction listed with GenBank Accession No. NG_016607.1; anucleic acid sequence corresponding to a region of interest for any oneof the exons 1-10 described herein for GenBank Accession No. NG_007111.1or SEQ ID NO: 33, or to an intron-exon junction, or to an exon-intronjunction listed with GenBank Accession No. NG_007111.1 or SEQ ID NO: 33;a nucleic acid sequence corresponding to a region of interest for anyone of the exons 1-19 described herein for GenBank Accession No.NG_007109.2, or to an intron-exon junction, or to an exon-intronjunction listed with GenBank Accession No. NG_007109.2; a nucleic acidsequence corresponding to a region of interest for any one of the exons1-13 described herein for GenBank Accession No. NG_008648.1, or to anintron-exon junction, or to an exon-intron junction listed with GenBankAccession No. NG_008648.1; a nucleic acid sequence corresponding to aregion of interest for any one of the exons 1-13 described herein forGenBank Accession No. NG_008649.1 or SEQ ID NO: 1, or to an intron-exonjunction, or to an exon-intron junction listed with GenBank AccessionNo. NG_008649.1 or SEQ ID NO: 1; or a nucleic acid sequencecorresponding to a region of interest for any one of the exons 1-15described herein for GenBank Accession No. NG_008466.1, or to anintron-exon junction, or to an exon-intron junction listed with GenBankAccession No. NG_008466.1. In one embodiment, the DRED is selected fromthose diseases listed in Table 1 or Table 2.

An aspect of the invention provides for a kit for monitoring theefficacy of treatment of a DNA Repeat Expansion Disease (DRED) in asubject. In one embodiment, the kit comprises at least one primer andinstructions for use. In one embodiment, the kit comprises a secondprimer. In one embodiment, the primer comprises a nucleic acid sequencehaving at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% identity to a nucleic acidsequence comprising SEQ ID NO: 5. In one embodiment, the primercomprises a nucleic acid sequence having at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identity to a nucleic acid sequence comprising SEQ ID NO: 29. Inone embodiment, the primer comprises a nucleic acid sequence having atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identity to a nucleic acid sequencecomprising SEQ ID NO: 30. In one embodiment, the primer comprises anucleic acid sequence having at least 80%, at least 85%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% identity toa nucleic acid sequence comprising SEQ ID NO: 6. In one embodiment, theprimer comprises a nucleic acid sequence having at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% identity to a nucleic acid sequence comprising SEQ ID NO:31. In one embodiment, the primer comprises a nucleic acid sequencehaving at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% identity to a nucleic acidsequence comprising SEQ ID NO: 32. In one embodiment, the DRED isselected from those diseases listed in Table 1 or Table 2.

An aspect of the invention also provides for a kit for monitoring theprogression of a DNA Repeat Expansion Disease (DRED). In one embodiment,the kit comprises a primer directed to a complementary target nucleicacid sequence of a gene or gene product encoding MLH3 and instructionsfor use. In one embodiment, the kit comprises a second primer. In oneembodiment, the primer comprises a nucleic acid sequence having at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% identity to a nucleic acid sequencecomprising SEQ ID NO: 5. In one embodiment, the primer comprises anucleic acid sequence having at least 80%, at least 85%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% identity toa nucleic acid sequence comprising SEQ ID NO: 29. In one embodiment, theprimer comprises a nucleic acid sequence having at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% identity to a nucleic acid sequence comprising SEQ ID NO:30. In one embodiment, the primer comprises a nucleic acid sequencehaving at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% identity to a nucleic acidsequence comprising SEQ ID NO: 6. In one embodiment, the primercomprises a nucleic acid sequence having at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identity to a nucleic acid sequence comprising SEQ ID NO: 31. Inone embodiment, the primer comprises a nucleic acid sequence having atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identity to a nucleic acid sequencecomprising SEQ ID NO: 32. In one embodiment, the DRED is selected fromthose diseases listed in Table 1 or Table 2.

An aspect of the invention provides for a method for treating a DNARepeat Expansion Disease (DRED) in a subject in need thereof. In oneembodiment, the method comprises administering to the subject aneffective amount of a nuclease-resistant oligonucleotide compound asdescribed herein. In one embodiment, the DRED is selected from thosediseases listed in Table 1 or Table 2.

An aspect of the invention provides for a method for treating a DNARepeat Expansion Disease (DRED) in a subject in need thereof. In oneembodiment, the method comprises administering to the subject aneffective amount of a pharmaceutical composition comprising anuclease-resistant oligonucleotide compound as described herein. In oneembodiment, the DRED is selected from those diseases listed in Table 1or Table 2.

An aspect of the invention provides for a method for treating a DNARepeat Expansion Disease (DRED) in a subject in need thereof wherein themethod comprises administering to the subject an effective amount of anoligonucleotide complex described herein. In one embodiment, the DRED isselected from those diseases listed in Table 1 or Table 2.

An aspect of the invention provides for a method for treating a subjectin need comprising administering a nuclease-resistant oligonucleotidecompound that promotes the skipping of region(s) of a gene product. Forexample, the oligonucleotide comprises a sequence that specificallyhybridizes in a human cell with a nucleic acid sequence encoding asubunit of the MMR system (e.g., MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, orPMS2). In one embodiment, the nucleic acid sequence is a complementarysequence for a human MLH3 gene or gene product. In one embodiment, thenuclease-resistant oligonucleotide is useful for inducing exon skipping.For example, oligonucleotide(s) can induce skipping of MSH2 exon 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or a combination ofMSH2 exons. Oligonucleotide(s) can induce skipping of MSH3 exon 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or a combination of MSH3 exons. Oligonucleotide(s) can induceskipping of MSH6 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or a combination ofMSH6 exons. Oligonucleotide(s) can induce skipping of MLH1 exon 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or acombination of MLH1 exons. Oligonucleotide(s) can induce skipping ofMLH3 exon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or a combination ofMLH3 exons. Oligonucleotide(s) can induce skipping of PMS1 exon 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or a combination of PMS1 exons.Oligonucleotide(s) can induce skipping of PMS2 exon 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, or a combination of PMS2 exons. In oneembodiment, the oligonucleotide directs skipping of one or more exons ofMSH2, MSH3, MSH6, PMS1, PMS2, MLH1, or MLH3. In one embodiment, one ormore oligonucleotides can induce skipping of MLH3 exon 7. In oneembodiment, the oligonucleotide is decreases the rate of DNA repeatexpansion. In one embodiment, the oligonucleotide is useful in treatinga DNA repeat expansion disease. In one embodiment, the target nucleicacid sequence is located on human chromosome 2, 3, 5, 7, or 14. In oneembodiment, the target nucleic acid sequence (or target complementarynucleic acid sequence) comprises: a nucleic acid sequence correspondingto a region of interest for any one of the exons 1-16 described hereinfor GenBank Accession No. NG_007110.2, or to an intron-exon junction, orto an exon-intron junction listed with GenBank Accession No.NG_007110.2; a nucleic acid sequence corresponding to a region ofinterest for any one of the exons 1-24 described herein for GenBankAccession No. NG_016607.1, or to an intron-exon junction, or to anexon-intron junction listed with GenBank Accession No. NG_016607.1; anucleic acid sequence corresponding to a region of interest for any oneof the exons 1-10 described herein for GenBank Accession No. NG_007111.1or SEQ ID NO: 33, or to an intron-exon junction, or to an exon-intronjunction listed with GenBank Accession No. NG_007111.1 or SEQ ID NO: 33;a nucleic acid sequence corresponding to a region of interest for anyone of the exons 1-19 described herein for GenBank Accession No.NG_007109.2, or to an intron-exon junction, or to an exon-intronjunction listed with GenBank Accession No. NG_007109.2; a nucleic acidsequence corresponding to a region of interest for any one of the exons1-13 described herein for GenBank Accession No. NG_008648.1, or to anintron-exon junction, or to an exon-intron junction listed with GenBankAccession No. NG_008648.1; a nucleic acid sequence corresponding to aregion of interest for any one of the exons 1-13 described herein forGenBank Accession No. NG_008649.1 or SEQ ID NO: 1, or to an intron-exonjunction, or to an exon-intron junction listed with GenBank AccessionNo. NG_008649.1 or SEQ ID NO: 1; or a nucleic acid sequencecorresponding to a region of interest for any one of the exons 1-15described herein for GenBank Accession No. NG_008466.1, or to anintron-exon junction, or to an exon-intron junction listed with GenBankAccession No. NG_008466.1. In one embodiment, the component of the MMRcomplex comprises MutS or MutL. In one embodiment, MutS comprises asubunit selected from the group consisting of MSH2, MSH3, and MSH6. Inone embodiment, MutL comprises a subunit selected from the groupconsisting of MLH1, MLH3, PMS1, and PMS2. In one embodiment, MLH3comprises SEQ ID NO: 1. In one embodiment, the oligonucleotidehybridizes to the target complementary nucleic acid sequence comprisingSEQ ID NO: 2. In one embodiment, the oligonucleotide has at least 60%,at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% identity toa nucleic acid sequence comprising SEQ ID NO: 3 or SEQ ID NO: 4. In oneembodiment, the oligonucleotide has at least 60%, at least 65%, at least70%, at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% identity to a nucleic acidsequence depicted in Table 4. In one embodiment, the oligonucleotidecomprises 15 to 30 nucleotide bases in length. In one embodiment, theoligonucleotide comprises one or more morpholino subunits, one or morelocked nucleic acid subunits, one or more 2-O-methyl moieties, or one ormore peptide moieties. In one embodiment, the subject in need isdiagnosed with a repeat expansion disorder (e.g., a DRED). In oneembodiment, the DRED is selected from those diseases listed in Table 1or Table 2.

BRIEF DESCRIPTION OF THE FIGURES

The patent of application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing (s) will be provided by the Office upon request andpayment of the necessary fee.

FIGS. 1A-F show a working model for transcription initiated DNA repeatexpansion via mismatch repair. FIG. 1A shows part of a GAA⋅TTC repeat(SEQ ID NO: 41) depicted with the purine (or R) strand in red, and thepyrimidine (or Y) strand in yellow. The numbered bases show alignment inregister. FIG. 1B shows that during transcription, the two strands areseparated and a variety of structures can form. One likely example, anRNA⋅DNA hybrid, is shown. FIG. 1C shows resolution of a structure thatcan lead to an out-of-register re-annealing within the repeat. Figurediscloses SEQ ID NOS 42 and 43, respectively, in order of appearance.FIG. 1D shows the small loop that is formed becomes a target for bindingby mismatch repair complex, MutSβ. Figure discloses SEQ ID NO: 44. FIG.1E depicts MutSβ in turn recruiting MutLγ, an endonuclease. Figurediscloses SEQ ID NOS 44 and 43, respectively, in order of appearance.FIG. 1F shows that repeat expansion has occurred with the addition oftwo trinucleotides (*) after repair initiated by MutSβ and facilitatedby MutLγ. Figure discloses SEQ ID NO: 42.

FIGS. 2A-B show the knockdown of MLH1 or MLH3 significantly reducesGAA⋅TTC expansion rate in FRDA model cells. Four independent lines weretransduced with the indicated knockdown lentiviral pools. FIG. 2A is arepresentative gel image of PCR products measuring GAA⋅TTC lengths. FIG.2B is a bar graph showing mean expansion rates. Compared to the emptyvector control virus (pLKO) MLH1sh was significantly different(p=0.0009) as was MLH3sh (p=0.00045) whereas PMS2sh did not reachsignificance (p=0.053).

FIG. 3 shows that MLH3 knockdown does not affect MutLα expression.Western blot probed for PMS2, MLH1 and ACTB (β-actin) shows long-termlentiviral-mediated shRNA knockdown of MLH1, PMS2 and MLH3 protein inthe cells used for DNA in FIGS. 2A-B. Knockdown of MLH1 concurrentlydepletes PMS2 (lanes MLH1sh). Knockdown of PMS2 halves MLH1 levels(lanes PMS2sh). Knockdown of MLH3 has a little effect on MLH1 proteinlevels, and no effect on PMS2 levels (lanes MLH3sh). None of the MLH3antibodies tried were effective for western blots.

FIG. 4 is a schematic showing that human MLH3, a component of MutLg has2 isoforms. MLH3 isoform 1 includes exon 7, which contains a highlyconserved portion of an endonuclease domain, while MLH3 isoform 2 lacksthis 72 base exon. Excluding exon 7 would approximate a functional knockout if the endonuclease activity of MLH3 is critical to repeatexpansion.

FIG. 5 is a schematic showing that splice switching oligos (SSOs) weredesigned to bind the acceptor or donor region of MLH3 exon 7 inpre-mRNA. These SSOs were used to induce skipping of exon 7 andpreferential production of MLH3 splice isoform 2 (iso2).

FIG. 6 is a photographic image of a gel showing that RT-PCR demonstratesexon skipping in MLH3 mRNA from SSO treated cells. Acceptor, donor orboth SSOs were given twice a week to FRDA rapid expansion model cells inculture. Cells were assessed for MLH3 isoform expression with RT-PCR.The combination of acceptor and donor SSOs at 500 nM effectivelyexcluded exon 7 as shown in lane 6 (arrow). M: Alkb plus size standardshowing 650 bp, 500 bp, 400 bp, 300 bp and 200 bp.

FIG. 7 is a photographic image of a PCR analytic gel showing thatpreferential expression of MLH3 isoform 2 leads to slower GAA⋅TTC repeatexpansion. PCR analysis of GAA⋅TTC expansion at week 3 with indicatedtreatments. Repeat size assay PCR product equals 500 bp flankingsequence+3×(repeats). Sample 6 with 250 nM each of both acceptor anddonor SSOs slowed expansion (arrow). M: Alkb plus size standard showing3,000 bp, 2,000 bp, 1,650 bp, and 1,000 bp.

FIG. 8 is a photographic image of a gel showing that single treatmentwith MLH3 SSOs slows expansion in non-dividing FRDA cells. PCR productsshow long (L) and short (S) alleles from FRDA patient fibroblasts.

FIG. 9A is a photographic image of gel showing detection of MLH3isoforms 1 and 2. Defined templates containing or excluding MLH3 exon 7were mixed in 10:1, 1:1 and 1:10 ratios. Primer pairs, MLH3 L3324 andMLH3 R3757 detected MLH3 isoforms 1 and 2. M: A 1 kb plus size standardshowing 500 bp, 400 bp, and 300 bp.

FIG. 9B is a schematic showing that MLH3, a component of a MutL complex,has 2 isoforms due to alternative splicing. MLH3 iso1 has exon 7 andMLH3 iso2 lacks exon 7.

FIG. 10 is a schematic illustrating that MLH3 iso1 is required forexpansion by following expansion in a human cell model. Single genomicconstruct, rather than 2 alleles simplifies analysis. Because theGAA⋅TTC repeat is not in the FXN gene in the model cell, repeatexpansion is freed from the negative effect of insufficient FXN. Primerstargeted for unique flanking sequences. GAA⋅TTC repeats expandincrementally and continuously in these model cells.

FIG. 11 is a UCSC Genome Brower image illustrating concordance of mouseand human MLH3 gene structures. The red oval indicates the variable exonin hMLH3 that is missing in hMLH3 isoform 2. This variable exon containsa conserved endonuclease domain.

FIG. 12 is a schematic illustrating that splice switchingoligonucleotides target a subset of mMLH3 exons. SSOs function bytightly binding pre-mRNA at splice junctions thereby excluding splicingfactors.

FIG. 13 shows individual and paired SSO activity in mouse Neuro-2Acells. Cultured cells treated with the indicated morpholino oligomers atthe indicated concentrations were lysed after 48 hours and RNA wasisolated for cDNA synthesis. PCR analysis of the cDNA usingoligonucleotides flanking the region of interest in the mMlh3 mRNAresulted in specific bands corresponding to no skipping (720 bp),skipping exon 5 or exon 6 (˜648 bp), or skipping both exons 5 and 6 (575bp). Lane M is the 1 Kb plus DNA ladder showing bands of 1000, 850, 650,500 and 400 base pairs.

FIG. 14 shows Mlh3 exon skipping is evident in adult mice 48 hours aftera single injection. Adult C57BL/6J mice were given a single dose (5μg/g) of a mixture of mMlh3ac5 and mMlh3dr7 cell penetrating “vivo”morpholino SSOs in the tail vein. Tissues were collected after 48 hoursand RNA was isolated to identify the splice variants. In thisexperiment, 3 of 4 mice injected with SSOs exhibited the desired mMlh3exon skipping in kidney (green arrows). Controls (C) were injected withsaline.

FIG. 15 shows Mlh3 exon skipping is robust in adult mice 48 hours aftera single high dose injection. Adult C57BL/6J mice were given a singledose (50 μg/g) of a mixture of mMlh3ac5 and mMlh3dr7 cell penetrating“vivo” morpholino SSOs in the tail vein. Tissues were collected after 48hours and RNA was isolated to identify the splice variants. In thisexperiment, 2 of 2 mice injected with SSOs exhibited robust mMlh3 exonskipping in kidney (green arrows). In these mice the treatment did notpenetrate the blood brain barrier and mMlh3 remained as isoform 1 inbrain (Br).

DETAILED DESCRIPTION OF THE INVENTION Abbreviations and Definitions

The singular forms “a”, “an” and “the” include plural reference unlessthe context clearly dictates otherwise. The use of the word “a” or “an”when used in conjunction with the term “comprising” in the claims and/orthe specification may mean “one,” but it is also consistent with themeaning of “one or more,” “at least one,” and “one or more than one.”

As used herein the term “about” is used herein to mean approximately,roughly, around, or in the region of. When the term “about” is used inconjunction with a numerical range, it modifies that range by extendingthe boundaries above and below the numerical values set forth. Ingeneral, the term “about” is used herein to modify a numerical valueabove and below the stated value by a variance of 20 percent up or down(higher or lower).

The terms “animal,” “subject,” and “patient” as used herein includes allmembers of the animal kingdom including, but not limited to, mammals,animals (e.g., cats, dogs, horses, swine, etc.) and humans.

Repeat Expansion Diseases

DNA repeat expansion disorders are a family of genetic disorderscharacterized by the pathogenic expansion of a repeat region within agenomic region (1,2). In such disorders, the number of repeats exceedsthat of a gene's normal, stable threshold, expanding into a diseasedrange. In most cases, the length of repeat expansion is negativelycorrelated with prognosis, i.e. longer repeats are correlated with anearlier age of onset and worsened disease severity. DNA repeat expansiondisorders are often called trinucleotide repeat (TNR) expansiondisorders because trinucleotide based disorders were the firstdiscovered and are the most widely known form of expansion diseases.However, expansions of up to twelve base repeat units have been found tocause disease (see Table 1).

TABLE 1 Repeat Expansion Diseases Disease, repeat Estimatedand gene affected Prevalence  1 Friedreich Ataxia (FRDA) 2-4/100,000GAA expansion in FXN (~1% of European descent carry)  2Fragile X Syndrome (FXS) 14/100,000 males CGG expansion in FMR1  3Huntington's Disease (HD) 2.71/100,000 CAG expansion in HTT  4Amyotrophic lateral 24%-46% of sclerosis/frontotemporal Familial ALSdementia (ALS/FTD) CCGGGG expansion in C9orf72  5Myotonic dystrophy type 1 5/100,000 (DM1) CTG expansion in DMPK  6Myotonic dystrophy type 2 12.5/100,000 (DM2) CCTG expansion in ZNF9  7Spinal & Bulbar muscular  0.75/100,000 atrophy (SBMA) CAG malesexpansion in AR  8 Spinocerebellar Ataxia 1/ 1.5/100,000SCA1 CAG expansion in ATXN1  9 SCA2 ? CAG expansion in ATXN2 10SCA3/Machado-Joseph ? disease (MJD) CAG expansion in ATXN3 11 SCA60.31/100,000 CAG expansion in CACNA1A 12 SCA7 2% of SCA'sCAG expansion in ATXN7 13 SCA8 2-5% autosomal CAG expansion in ATXN8dominant ataxias 14 SCA10 ? ATTCT expansion in ATXN10 15 SCA12 ?CAG expansion in PPP2R2B 16 SCA17 ? CAG/CAA expansion in TBP 17 SCA31 ?TGGAA expansion in TK2 18 SCA36 ? GGCCTG in NOP56 19 Dentatorubral-0.48/100,000 pallidoluysian of Japanese atrophy (DRPLA) CAG populationexpansion in DRPLA 20 Oculopharyngeal muscular dystrophy (OPMD)CGG expansion in PABPN1 21 Progressive myoclonus 5/100,000epilepsy (EPM1) Finnish births CCCCGCCCCGCG (SEQ ID NO: 34)expansion in CSTB

The repeat expansion disorders that were discovered first arepredominantly dominant diseases, such as Huntington's disease. However,recessive DNA repeat expansion disorders are a rapidly growing subclass.For example, the progressive, neurodegenerative disease Friedreichataxia (FRDA) is caused by a repeat expansion in the FXN gene from thenormal range of 6 to 36 repeats to the diseased range of approximately600 to 1600 repeats (3). The GAA⋅TTC repeat expanded from an Alu elementin the FXN first intron (4,5). Disease severity correlates to the lengthof the expanded repeats and the consequent reduction of FXN geneexpression. Over a million Alu elements together constitute about 11% ofthe human genome (6), suggesting a vast reservoir for other suchexpansions. Indeed, the CCTG expansion responsible for Myotonicdystrophy type 2 (DM2) and the ATTCT expansion causing spinocerebellarataxia type 10 (SCA10) also expanded from Alu elements (7,8).

To effectively treat a relentlessly progressive and lethal disease likeFriedreich ataxia, the underlying DNA repeat expansion must beaddressed. Currently there is no effective treatment and no cure for anyof the DNA repeat expansion diseases (see Table 2). In one embodiment,the invention is directed to treatment of DNA repeat expansion diseases(DREDs) using oligonucleotide compositions discussed herein.

TABLE 2 Exemplary DNA repeat expansion diseases. Friedreich's AtaxiaSpinocerebellar ataxia type 3 Blepharophimosis-ptosis- Spinocerebellarataxia type 6 epicanthus inversus Cleidocranial dysplasiaSpinocerebellar ataxia type 7 Congenital central hypoventilationSpinocerebellar ataxia type 8 Dentatorubralpallidoluysian atrophySpinocerebellar ataxia type 10 Fragile X syndrome Spinocerebellar ataxiatype 12 FRAXE mental retardation Spinocerebellar ataxia type 17Hand-foot-genital Spincocerebellar ataxia type 31 HoloprosencephalySpinocerebellar ataxia type 36 Myoclonus epilepsy type 1 Huntington'sdisease like 2 Myotonic dystrophy type 1 Spinal and Bulbar Muscularatrophy Myotonic dystrophy type 2 Huntington's disease Oculopharyngealmuscular dystrophy Synpolydactyly Spinocerebellar ataxia type 1 FragileX-associated tremor/ataxia syndrome Spinocerebellar ataxia type 2Syndromic and nonsyndromic X-linked mental retardation AmyotyophicLateral Sclerosis/ Dentatorubral pallidoluysian Frontotemporal Dementiaatrophy Progressive myoclonus epilepsy

Sufficiently long DNA repeats such as those seen in DNA repeat expansiondisorders are characterized by genomic instability of the repeatedregion. Often, the repeated regions frequently change in length duringintergenerational transmission and within somatic cells. This fact holdsboth clinical and emotional relevance. Clinically, longer repeat lengthsare associated with increased disease severity and earlier age of onset.Additionally, the progressive repeat expansion may actually causedevelopment of the disease that otherwise would not develop, as theexpansion of only 1 additional repeat can result in an individual'srepeat region expanding into the diseased range. Emotionally, thispossibility may result in increased anxiety and depression in the ‘atrisk’ individual. Unfortunately, there are no therapeutics to slowdisease repeat expansion.

DNA Mismatch Repair (MMR)

The molecular mechanism underlying repeat expansion largely remainsunclear. Without being bound by theory, DNA mismatch repair contributesto the genomic instability observed in trinucleotide repeat expansiondisorders. DNA mismatch repair (MMR) is a pathway that normallyrecognizes and repairs DNA errors made during replication. However, whenit comes to certain types of repetitive DNA, MMR actually can contributeto genome instability. For example, a contribution by the MMR pathwayhas been established in several repeat expansion diseases includingmyotonic dystrophy, Huntington's disease, and Friedreich ataxia. Forexample, the inventors' laboratory has contributed to the understandingof the role of MMR in Friedreich ataxia. MMR requires the sequentialaction of the protein complexes MutS and then MutL. Humans have twodifferent MutS complexes, alpha and beta, and three different MutLcomplexes, alpha, beta, and gamma. In human cells, DNA mismatches areinitially recognized by a MutS protein heterodimer prior to recruitmentof a MutL complex. MutSalpha, a heterodimer of MSH2 and MSH6, is thedominant MutS complex that recognizes base-base mismatches and shortinsertion/deletion loops. MutSbeta, a complex of MSH2 and MSH3, is lessabundant than MutSalpha in most cell types, and appears to befunctionally redundant to MutSalpha. In MMR, MutS heterodimers recognizea mismatch but a MutL heterodimer is required as the next step in themismatch repair process. In humans there are four identified MutLhomologues: MLH1, MLH3, PMS1 and PMS2 (9-12). MLH1 is the mastersubunit, much like MSH2 in the MutS system. MLH1 combines with PMS2 toform MutLalpha, with PMS1 to form MutLbeta and with MLH3 to formMutLgamma, respectively. Also like MSH2 and its partners, MLH1 and itspartners are more stable as heterodimers (11). MutLalpha is the dominantspecies, being about ten-fold more abundant than MutLbeta (11), similarto the ratio between MutSalpha and MutSbeta (13). MutLgamma is lessabundant still: about 2% the level of MutLalpha (14). MLH3 expressionlevels are considerably lower than the other binding partners of MLH1and MLH3 is currently considered to be a minor player in MMR processes,as it has mostly redundant functions. Without being bound by theory,MLH3, while a minor player in canonical MMR, is a major force in DNArepeat expansion. In one embodiment, the present invention utilizesoligonucleotides (for example, antisense oligonucleotides resistant tonuclease digestion), for use in modulating the expression and/orfunction of subunits of the MMR system that are encoded by nucleic acidmolecules discussed herein, ultimately modulating the amount of a MMRsystem subunit that is expressed and/or produced. In one embodiment, theMMR system subunit comprises MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, orPMS2. In one embodiment, the MMR system subunit comprises MSH2, MSH3, orMSH6. In one embodiment, the MMR system subunit comprises MLH1, MLH3,PMS1, or PMS2. In one embodiment, the he MMR system subunit comprisesMLH1 or MLH3. In one embodiment, the MMR system subunit comprises MLH1.In one embodiment, the MMR system subunit comprises MLH3. In oneembodiment, the MMR system subunit comprises PMS1. In one embodiment,the MMR system subunit comprises PMS2. In one embodiment, the MMR systemsubunit comprises MSH2. In one embodiment, the MMR system subunitcomprises MSH3. In one embodiment, the MMR system subunit comprisesMSH6. In one embodiment, the expression and/or function of a subunit ofthe MMR system is decreased.

The genomic sequence for human MSH2 (found on human chromosome 2) hasGenBank Accession No. NG_007110.2. Sequence information related to humanMSH2 (isoform 1) is accessible in public databases by GenBank Accessionnumbers NP_000242.1 (protein) and NM 000251.2 (nucleic acid). Sequenceinformation related to human MSH2 (isoform 2) is accessible in publicdatabases by GenBank Accession numbers NP_001245210.1 (protein) andNM_001258281.1 (nucleic acid). The genomic sequence for human MSH2(GenBank Accession No. NG_007110.2; 166,188 bp in length), is found atnucleotide no. 4,944 and terminates at nucleotide no. 85,105, whereinexon 1 is located between nucleotides 4,944 and 5,279; exon 2 is locatedbetween nucleotides 10,278 and 10,432; exon 3 is located betweennucleotides 11,971 and 12,249; exon 4 is located between nucleotides14,291 and 14,437; exon 5 is located between nucleotides 16,146 and16,295; exon 6 is located between nucleotides 18,173 and 18,306; exon 7is located between nucleotides 31,619 and 31,818; exon 8 is locatedbetween nucleotides 47,425 and 47,534; exon 9 is located betweennucleotides 64,908 and 65,031; exon 10 is located between nucleotides68,535 and 68,685; exon 11 is located between nucleotides 72,842 and72,939; exon 12 is located between nucleotides 76,902 and 77,147; exon13 is located between nucleotides 78,244 and 78,448; exon 14 is locatedbetween nucleotides 80,149 and 80,396; exon 15 is located betweennucleotides 82,573 and 82,748; and exon 16 is located betweennucleotides 84,656 and 85,105. It is understood that intron sequenceprecedes and follows the denoted nucleotide regions comprising the exonsequences listed herein. Oligonucleotide compounds (e.g., exon skippingor intron retaining SSOs) can be directed to the nucleic acid sequencecorresponding to the region of interest for each of the exons 1-16described herein, and intron-exon junctions, or exon-intron junctionslisted with GenBank Accession No. NG_007110.2.

The genomic sequence for human MSH3 (found on human chromosome 5) hasGenBank Accession No. NG_016607.1. Sequence information related to humanMSH3 is accessible in public databases by GenBank Accession numbersNP_002430.3 (protein) and NM_002439.4 (nucleic acid). The genomicsequence for human MSH3 (GenBank Accession No. NG_016607.1; 229,341 bpin length), is found at nucleotide no. 5,174 and terminates atnucleotide no. 227,341, wherein exon 1 is located between nucleotides5,174 and 5,490; exon 2 is located between nucleotides 6,937 and 7,057;exon 3 is located between nucleotides 15,669 and 15,889; exon 4 islocated between nucleotides 20,623 and 20,835; exon 5 is located betweennucleotides 22,770 and 22,886; exon 6 is located between nucleotides23,267 and 23,384; exon 7 is located between nucleotides 25,509 and25,654; exon 8 is located between nucleotides 29,453 and 29,619; exon 9is located between nucleotides 75,979 and 76,091; exon 10 is locatedbetween nucleotides 79,377 and 79,491; exon 11 is located betweennucleotides 91,990 and 92,074; exon 12 is located between nucleotides95,032 and 95,141; exon 13 is located between nucleotides 112,072 and112,204; exon 14 is located between nucleotides 118,459 and 118,646;exon 15 is located between nucleotides 119,361 and 119,529; exon 16 islocated between nucleotides 126,220 and 126,284; exon 17 is locatedbetween nucleotides 129,246 and 129,362; exon 18 is located betweennucleotides 138,091 and 138,198; exon 19 is located between nucleotides143,259 and 143,370; exon 20 is located between nucleotides 164,110 and164,267; exon 21 is located between nucleotides 204,656 and 204,842;exon 22 is located between nucleotides 215,339 and 215,468; exon 23 islocated between nucleotides 223,642 and 223,813; and exon 24 is locatedbetween nucleotides 226,277 and 237,341. It is understood that intronsequence precedes and follows the denoted nucleotide regions comprisingthe exon sequences listed herein. Oligonucleotide compounds (e.g., exonskipping or intron retaining SSOs) can be directed to the nucleic acidsequence corresponding to the region of interest for each of the exons1-24 described herein, and intron-exon junctions, or exon-intronjunctions listed with GenBank Accession No. NG_016607.1.

The genomic sequence for human MSH6 (found on human chromosome 2) hasGenBank Accession No. NG_007111.1. Sequence information related to humanMSH6 (isoform 1) is accessible in public databases by GenBank Accessionnumbers NP_000170.1 (protein) and NM_000179.2 (nucleic acid). Sequenceinformation related to human MSH6 (isoform 2) is accessible in publicdatabases by GenBank Accession numbers NP_001268421.1 (protein) andNM_001281492.1 (nucleic acid). Sequence information related to humanMSH6 (isoform 3) is accessible in public databases by GenBank Accessionnumbers NP_001268422.1 (protein) and NM_001281493.1 (nucleic acid). Thegenomic sequence for human MSH6 (GenBank Accession No. NG_007111.1;30,807 bp in length; SEQ ID NO: 33), is found at nucleotide no. 4,936and terminates at nucleotide no. 28,807, wherein exon 1 is locatedbetween nucleotides 4,936 and 5,347; exon 2 is located betweennucleotides 12,781 and 12,977; exon 3 is located between nucleotides17,748 and 17,917; exon 4 is located between nucleotides 20,465 and23,009; exon 5 is located between nucleotides 25,274 and 25,539; exon 6is located between nucleotides 26,764 and 26,881; exon 7 is locatedbetween nucleotides 27,472 and 27,561; exon 8 is located betweennucleotides 28,058 and 28,212; exon 9 is located between nucleotides28,306 and 28,505; and exon 10 is located between nucleotides 28,633 and28,807. It is understood that intron sequence precedes and follows thedenoted nucleotide regions comprising the exon sequences listed herein.Oligonucleotide compounds (e.g., exon skipping or intron retaining SSOs)can be directed to the nucleic acid sequence corresponding to the regionof interest for each of the exons 1-10 described herein, and intron-exonjunctions, or exon-intron junctions listed with GenBank Accession No.NG_007111.1 or described in SEQ ID NO: 33.

SEQ ID NO: 33 (exon sequences are highlighted and bolded therein): 1cactatgttg ctagctggtc ttgaactcct ggcctcaagt catccttctg tcttggcctc 61ccaaagtgtt gggattgtaa gtgtgagcca ctgtccctgg ccagttggtg atttatttgt 121ataactgtcc aatttattga atacgtatgg cgtgccaagc actgggctga gggcttcata 181atgccctttc actcaatgct tagcacaacc catgaagaag gtagtgttaa tatcatccct 241gttttacaga tgtagaaact gaggcacagg ctaaataact tgcccaacaa gctcgtgcag 301tttagtaagc agctcagctg ggatgtgaac acaaactttg aatacagagc tcttaaccag 361taggccagag gttcccaaac acctagtata tttactacct tagtactttc ctgccacatc 421tcctaggcca aaacaactcc attgattgtt atgcaattta ctaagtgtag ccatttgaaa 481aaaaaataca tataaaagaa aaatattttt attcagtttt caaaataacc catatatagt 541catggaatgt atgtgttggt tgggcactgc agactaaaga cagtttaggg ccgggcacgg 601tggctcaggc ctgtaatcct agcactttgg gaggccaagt agagagaagg gcttgagccc 661aagagttgga gaccggcctg ggcaacatag caagacccag tctctacaga aaataaaatt 721atcagggtgt gaggacgcac acctgcagtc ttagctgctt gggaggctga ggctggagga 781tcagcctggg caacagagtg agaccctgtc tcagaaaaaa aaaaaaaaaa agacagactt 841ttattcagat atgcatgcag gagttcacag aaaaaaaaag tgagtccagg aggctgttat 901ttggcattta tacaactttt tttttcttga atctcgaaat ctactttata tataccattt 961aataggggaa gaggagggag aaaaagcctt ccatgggaag aacaaatagg tttctggggg 1021aacaaaaggg agataagaat gtttgttttt gcaggtgcaa gtggtctttg tctttttttc 1081tggccactaa aactccccta gagaggagat ttacggcagc ttcactccca gaaatttctg 1141ctgttagtcg cataagggaa gctttgaaac ggcatctttc tgcatctgtt ggctctcaaa 1201tgtcttcagt tccaagtaac attcatgcca attctggggg tctgagtgtc cccacataat 1261acatgtgttc tcttgtcttt taatgaagtt tgtgggaggc atctaactgt agcctccaaa 1321atttggccca taggtactac tgtccttatc aaagacgagg aaacaagttc agaaaagtat 1381taattgctcc gagttatctg cttggctagc taggatcaga gctcagttct ccatttaacc 1441caaagcccag gctcttaacc tcttacaact ggcgcatccc ctctgaacct ccatttcctc 1501cctgtaaaag aataacatcg gccgggcgca gtggctcaca tctataatcc cagcactttg 1561ggaggcagag atgggcggat cacgaggtca ggagtttgag accagcctgg ccaacatggt 1621gaaaccccat ctctactaaa aatacaaaac ttagctgggt gtgttggtgc ctgtaacccc 1681agctactcag gagactgagg caggagaatt gccttaacct gggaggcgga ggttgtggtg 1741agccaagatc gtgccattgc actccagcct cggtgacaga gcaagactcc atccccaaaa 1801aaacaaacaa caacaacaaa aagagaataa cgttatattc agttgaacca aaatgaatta 1861aatattaata tttgtacttc aaaaacggtc cagcttggct gggcgcagtg gctcccgcct 1921gtaatcccaa cattttggga ggccgaggca ggaggatcat ttgaggtcag gagtttgaga 1981ccagcctggc caacatggtg aaatcctgtc tctactaaaa atacaaaaat tagctgggca 2041gtagtagcgc gtgccggtaa tcccagctat tcaggaggct gaggaaggag aattgcttga 2101gcttgggagg tgaaagttgt ggtgagctga gactgcacta ctgcacacca gtctgggaga 2161cagagtaaga ccctgtctca aaacaaaaca accaaaaaac caaaaaggtc cagcttgggc 2221aacatagtga aacttcgtct ctacagaaaa tttttaaaat actagcaggg caccgggcac 2281agtggctcat acctgtaatc ccagcacttt gggaggctga ggcaggcggg tcacttgtgg 2341tcaggagttt gggatcaggc aggccaacat ggtgaaaccg tgtctctact aaaaaacaaa 2401aattagctgg gcatggtggt aggcaccagt aatcctagca ctcaggaggc tgaggcatga 2461gaattgcctg aacccgcaaa gcaggggttg cagtgaacca agatggcgtc actgtactcc 2521agcctgggtg acagaataag actcctcaat taaaaaaaaa aaaaattagc tgggcatggt 2581gttgcgggcc tgtggtccca ggtactcagg aggctgaggt gagaggatta cttaagcctg 2641ggaggttgag gctacagtaa gccaagatca cgccactata ctccagcctc tgtgacagag 2701ccagaccctg tctcaaaaaa attttaaaaa gggcaaattt tggcaatttc acatagttca 2761acctagtata aggtggttgt aataactaaa tgagataaaa tggtgttaaa ttggaagtat 2821tatagtattt ctgttaacaa catagggctc cagaaccagc ttccttgagt ttaaatccag 2881gctccaccac ttcctagcta tgcagtcatg ggcaagttac ttgacccaac tgtgcctcag 2941cttcatccat gatatggaga tacaggataa ccagcctctt acgtgcaatt ctgaaatcca 3001aaaagctctg taaaccaaaa gtttgggggt aaactcattt ggtagcaaat tttgacctga 3061ggctatttat agtctatatt ctgtattctt tctacttagt atgaataagc atgtaagttt 3121tactgcatgt ttgatttcag catgttcccc cagactctct gggggtgttt acgtatgccg 3181gtgggggaaa gagaccaact ctcaaatatt atctcaaaca gttggtttca ctgtgcttgc 3241ttgggtagca catataccaa aattggaatg acccctgcac agggatgaaa tgcaaattcg 3301tgaagcatac tgtatttttc ttagcacata ccacctttgg caatattctt tttttttttt 3361tgagagggag tcttgctctg tcgcccaggc tggagtgcag aggcgcgatc tcggctcact 3421gcaagctccg cctcccgggt tcacaccatt ctcctacctc agcctcccca gtagctggga 3481ctacaggcgt gtgctaccac gccaggctaa ttttttgtat ttttagtaga ggcggggttt 3541cactgtgtta gccaggatgg tctcgatctc ctgacctcgt gatccgccca cctccgcccc 3601ccccccgaag tgccgagtgc tgggactaca ggcgtgagcc actgcgcccg gcccccgcct 3661ttttttttta gattgatttt attacttgcc tagcaaagga gaaccttctg gcagaacagt 3721ctccaagaac aaggcaaaca actaatttta cataggtttt taccaatgta cagctgttga 3781ttgtgactgg tttccggcaa tctggatttc acaatctgga taaggggaca aacaattgtc 3841tgtcttccac tatctttctt gaatttgaat agaacctttt tattctcata gcctcttagc 3901tttctttctt ttttttttga gacggagttt cgctcttgtc gcccaggctg gagtgcagtg 3961gcgcgacctt ggctcactgc aaacgctgcc tcccaggttc aagttattct cctgcctcag 4021cctcccaagt agctgggatt acaggcgcat gccaccacgc ccggctaatt tttggatttt 4081tagtagagac gggggtttca ccatgttgac taggctggtc ttcaacgcct gacctcaggt 4141gatccgcccg cctcggcatc ccaaagtgct gggattacag gcgtgagcca ctgcgcccgg 4201cctctcatag tctcttagct ttctaaaatt tgaaaaatcc tgtaaagaca cacctgggtc 4261aaagggctca gataacggac tgtggccctt aagtacttac gtcacaggtt attgagagga 4321tcgatttagt taccagatgt aaaatgctgg gatcagtgcc tggcaaagga aaactttgta 4381cagctgcagg ctttcaccat acacaacagc atcgctaacg aatgctatta caatattcat 4441ttagcgttta ccaagtgcct actctataca aatcttgaga atacaacgtg aaggtgaact 4501gctgactaaa gtttggtccc tttcgctccg tctccttgcg aaaatgctct aacggcagga 4561ggtcacgcga gcgctggacg cgtttctccc cgcgagcccc tttccgaggc ctttcgggtc 4621cccccggtta tccccgcccg ggcggtgcgc gcccccgctg ttcccgcttc cgctccagag 4681aggcagggct ttccgagcct gctagccccg cggccgcaac taaccccggg tcggagtgtt 4741ccggcccggc cagccccgcg gcgtgaggga aggggagctc agcagttccc cgcgcggggc 4801ccaggcgtcg gcggcagggc gggcccctca ccgccagcgt gccagccccg cccctaccca 4861ccagtgtgcc agccccgccc ttccccacgt cgccgcgcgc ccgggggcgg ggcctggcgc 4921

4981

5041

5101

5161

5221

5281

5341

5401 aacccggcga ggggaggctc gcacaggggg ttgggggggt gcacggcctg gccctgggct5461 cggaggaggc ggggccgcag agttggcttg aatgagtgca ggggtcgagt ctggagcatt5521 tgggggtgta gcttgtaaac agggtcggag gagagaggct gtgcaggaag agggctgcag5581 gggagacgcg gagagttcgg gccttttgga gggaggagac gcgtcccgcc aggtgggggt5641 gctgggctaa ggaaggggcg acgcgcgcag ctccgggtgg ggagggggcc tgggaggtgg5701 gagcactggg ggtggggcga gaaggggaag gcgcccggcc cacttggtgg gcggggcggg5761 gggcggggtg gcgggaagga ggaatgcctg cgggaggccg aacggggaga gtccggtggt5821 gtggggtgcg aaaggaggtt cctcggccgg cgcggagata gtgagttggg gctccagtag5881 tcgatcgagg tagacactta gaggtagtta agagccgcgg tcgccgagac gccttgggga5941 cggtgggcct tcggcctagg tgaggggccg ccgagggggt gggccacgag ctgcgagcgc6001 gggggggtgt gtcaccatgg ggaccgcggg gcctaattgg gcggggcggg gccgtgggga6061 gccgaagtgc tgggatccgg ctgggtcctt cggtaggtag gctgcacgtg caccgagacg6121 aagatagaat attttgacgt atgtggaaat tcgtgtcgag tggaaaatat tttattttat6181 gaaatagtgt aatttttatg gggcaccact gggcttttag aggccttaat cgggcgctgg6241 acaaagatgt gtggacgtga gtgactccgg ggaagcctgt cgggagttgt cctcacttta6301 tgggcagtta agtgcttttt tttttttttc ctttttgaga gagagtttcg ctcaagtcca6361 ggctggagtg caatggcgcg atctcagctc accgcaatct ccgcgtcccg gcttcaagcg6421 attccccagc ttcagcctcc cgagtagtcg ggattacagg aatgcgcccc cacaccccgc6481 caattttgta tttttagtag agacggggtt tctccatgtt ggtcaggcta gtctcggaat6541 tcccgacctc aggtgatcca cccgcctcgg cctcaaagtg ctgggattac aggcgctagc6601 caccgcgccc ggtctgttta gggcttttta tccgggcagc tggcgacatt ttgaaaagct6661 tgcttttgct gtttgccaga tacatatata tgtattttga gacagagtct tgctcttttg6721 tccaggctag agtgcagtgg cgcgttcttg gctcaccaca acctctgtct ctggatcaag6781 agattatcct gcctcagcct cccaagtagc tgggactaca ggtgcgcccc accacgcctg6841 gctaattttt gtatttttag tagagacggg tttcactatg ttggccaggc tggtatcgaa6901 ctcctgacct cttgatcggc ccgcattggc ctaccaaagt gctgggatta caggcatgaa6961 ccaccgagcc cggccgtttg tcagatacta aacacaaagt ttaatggtcg ctatttgaac7021 aaacgaagaa ataaaggctc agaaaaaata actcattcaa gataagagcc agttcgtgtt7081 ttttgtttgg ttttgttttg aaatggagtc tcgctctgtc gcccaggctg gagtgctgtg7141 gcgctttctc ggctcactgc aacctctgcc cgccgggttc aagtgattct cctgcctcag7201 cttcccgagt agctgggatt acgggtgtgc ccaccgcggt ccggctgatt tttcaccatg7261 gagtttcacc atgttggcca ggctggtctt gaaactgctg acctcaagtg gtccacccac7321 ttcagcctcc caaagtgctg ggattacagg tgtgagccac cgtgcccggc cgctagttag7381 tggttttgag taatggattt caaatccatt taaatccagt ttaaagtgtc ctaaaggaat7441 tctgagattt ttctaagtgt aattatagtg ttacccttgt ttaagcgacc ctttcccgca7501 gtttaaatat atatagttgt gcattagtag aatatgcttg tggggaacag agccagcatc7561 cgcaataaca aactcctggt tagaaaagca tgacgtattg tttacttgag catgaattga7621 ttgttgaatc caaaccaaac gggtgtattt attgtaagga tgtactttac attcatattg7681 aatagcgtat gttatttgtt tcttgaggtt gagtttaaga gacttgtaaa aataaaacgt7741 atacatttca cctcccgtta tggagaggat tccagggtat tcaagaaaga tgggcatttg7801 atactaggtt tctaaagaaa ctgcagtgtc tagatcactc tgccgagcac agcattaggc7861 attatggatc ctggatacaa ccatgaacag gacaaagcaa agaggcaatt gtagactcca7921 agtggaaagg ggacggagag gatgcgggtc aggctaggct ctcagctctg taaaccgaaa7981 ccagaaggac aaataagctt agacagatta tagtgagagt gggaagctgg ttcaggaaga8041 ggaaggtctg caaattgtgg gtaggatgaa aggaggagga gggagcattg gagaagttaa8101 gcagagatcc aatcatgaac agtctgatga gctacagaga cattcggact tactccatga8161 atcatttaag ccttaaaaca tgttgagcgt attttttttt tttttgagac ggaatttcac8221 tcttgttgcc caagctggag tgcagtggtg tggtctcagc tcactgcaac ctccgcctcc8281 tgggttccag cgattctcct gcctcatcct ctcaagtacc tggtattaca ggtgcctgcc8341 accacgccca gctaattttt gtgtttatag tagagacggg tttcaccatg ttggtcaggc8401 cagtcgtgaa ctcctgacct caggtgatcc acccacctca gcctcccaaa gtgttgggat8461 tacaggcgtg aaccaccgca cctggccgtg agccaccgtg tctgtccgag catcttttaa8521 tgtttgtcat ttagatttct tcttgtgctg aagtgtttgt cttttgctgt ttcttttttt8581 tttcctagtt ctttgtcatt tgtgtgtgat ataaatgtct tctttcacaa tgagttcttt8641 catttagttt atggctttgt tgttgttgtt gaataataga ggtctcactt tgttgcccag8701 gctggtgttg aactcttgct ctcaagcgat cctcccactt cagcctccca acctgttggg8761 attacaagtg tgagccacca cacccagcct tatggcatct ttcgatgaac aaattattga8821 ttataatgtg gaatttgtcc ttttattttc tctgtggtta gtgtttctat aggttttatt8881 taagaaatcc acagggaggc tgggtgcagt ggctcatgcc tgtaaaccca acactttggg8941 aggccaaggc aggccaacat ggctagaccc tgtctctcca aaaaataaga aaattagcca9001 ggcatggtgg cgtgtgcctg tagtcccagc ttcttgggag actgagatgg gaggatcgct9061 tgagtccagg aggttgaggc tgcagtaagc caagagatca tgccatgcac tccagcctgg9121 gtggcagagt cagaccctgt ctgccaaaaa ataaaataaa agttggtgaa aatgttgatt9181 atatatttta ggaacaacta gtaattgaca tcaaaattat gggctaaaga gaaagcaaaa9241 ataatgtgat tttaaaccag aattcaaaag atctgtttag cgtatgttta gacaaagcca9301 ttacttatta tatcaaagtt ttaacattta ttttgtgagc tgtcagcttt tcctcttaac9361 atttttcccc accgtcttaa aaaaccccaa gaataccgga catttaagac tcacttaaag9421 ctttaaaagc acttgcaaaa tcctaaaatc ataatttaag gtgtttttgg agggcaggag9481 caatggtggc aggcagtgtt ttgctttgtt gcccaggctg aagtacagtg gcagatctcg9541 gttcactgca ccctcgacct attcggctca agtgatcctc ccacctcagc tttctgagta9601 gctgggaccc caagtgcaca ccaccccatg cctggctaat ttttaaattt ttttgtagaa9661 acaaggtctc actgtgtagc ccagatggtc tcgaattcct gggctcttaa gagatcctcc9721 caaagtgctg ggatcatagg tgtgagccac cacacctggc ctattttggc attcttgaaa9781 accgcaggat taccacggat aaaattttaa aattaccttt aaagaattca ggtttacaca9841 caaaaaaaat ttggtttgtt agcagtgagt gaagaaaaat tttgagaaat gtttaaaatt9901 tttagttttg ttacacaata cattttacta cctgtttaat tatctttttt gactcagaaa9961 ccagtttcct gggtccagga tgtttagtgg tactcttttt cttcaagctt tttagcattg10021 gaggaactgc atattagtaa aatttttagt cttagcattt tatagcttac tgctatttct10081 tttctttcat tctttctttc tttttttttt tttttttttt ttttttgaga tggagtctcg10141 ccctgtcacc caggctggag tgcagtggca cgatctcggc ttactgcaac ctctgccttc10201 caggttcaaa tgattctcct gcttcagcct cccgagtagc tgggattaca gatgcccgcc10261 accatgccca gctaattttt atttttttag tagagatggg gtttcaccat gttggccagg10321 ccagtctcga actcctgacc tcgtgatcaa cccgccttgg cctttcaaag tgctgggatt10381 acaggcgtga gccaccgtgc ccagcctttt tctttttctt tttctttttt tttttttttt10441 tgagacggag tcttgctctg ttacccaggc tggagtgtag tggcatgatc tgggctcact10501 gcaacctcca cctcccgggt tcaagggagt ctcctgcttc agcctcccga gtagctggga10561 ttacaggcgc ctgccaccat gcccagctaa tttttgtatt tttttagtag agatggggtt10621 tcgccatgtt ggccaggctg gtcttgaact cctgacctca ggtgatctgc ctgcctcgtc10681 ctcccaaaat gctgggatta taggagtgag ccactgcgcc cggcccagca tactgctatt10741 tctttctttc tttcttcttc cttttttttt tttttgtttt tttttttttt tttttttttt10801 tgtgagacgg agtctgtcgt ccaggctgga atgcagtggc gttttcttgg ctcactgcaa10861 cctctgctgc ccgggttcaa gtgattctcc tgcttcaggc tcccaagtag ctgggattat10921 aggcctctgc cactgcactt ggctaatttt tgtatttttg gtagagacgg ggtttcacca10981 tcttggccag gctggtcttg aactcctgac ctcgtgatcc acctgccttg gcctcccaaa11041 gtgctgggat tacagacctg agccaccgca cccggcccat actgctattt cttaacagca11101 gagaaattat gtgtcagatt ctgtaagtgt aatggtatat aaaggataaa atgatgttga11161 aaaacaaaat tttttgttta aatgcttatg tttctaatat tttatttcag aaaggaattt11221 atttcaaaac tgataatggt tggatccagc ttttcacaca aacttttttt tcctagtgag11281 gatgcacatt tatcctgtaa acaaatggaa gacattattt ttttaattgc ttgcttagaa11341 atgaaataat tcttttctaa tgatctttta aagcatgaga cctcatacat catttaaaac11401 aatttatact gtattttaca catgacaaag ttctaaggta acagcccttt tctaagacta11461 aagttacagt cctccctttg tatctgaggg ggattggttg caggaccccc ctgtgaatac11521 ccaaatcctt ggatgtccaa gtcccttatg agatgtagta tttgcatata acctatacac11581 atcttcccct gtactttatc tctagattac gtacaatacc taatagaatg taaatgcttt11641 gaaattagtt gttcagctgt attttaaatt ttgtattttt tttccttttt ttttgagaca11701 gagtcttgct ctgttgccca ggctggagta cagtacagtg atcacagctc actgcacctt11761 taacctccca ggctcaagct gtcctgcctc ggcctcccca agtgttggga ttacaggtgt11821 gagccatcat acctggtcac tgttttttat tggttttaaa tttttgattt aaaattttta11881 atctaggttg gttgaatctg gactggaacc caaggatatg tttgttgagc atactgtatt11941 tactttggaa tacaactaga atgcttaact tgtatgttaa aaatacttta tttggccagg12001 cgcggtggct cacgcctgta atcccagcac tttgagaggc caaggcgggt gaatcatttg12061 aggtcaggag tttaagacga gcctggccaa catggcaaaa ccctgactct acaaaaaaaa12121 ggtaaaaata agccaggtgt gatggcgtgt gcctgtagtc ttggctattc aggaggctga12181 gacacaagaa tcgcttgaac cggggaggca cgttacgccc tcagttgttg acttgagttt12241 ttccgtagtt tgtaggggga gggtaataga gtattaggta gcttttggaa tacataggag12301 tgtaactgga aaaagattcc aagcaagtct aatgaattag ataatttacc taattagtaa12361 attatgtaat cagtatgctt tataataata ttgtgagtta gatcctgttt ctgatatgta12421 cataccatat tgtataggtg ctactaattt ggagagcata tacagtgagt ccatgccttt12481 ttcctgccat cagcattata ccaaaattct gccatggttt ttaaactttg attctgagaa12541 agtttctcac cctaataaca taactatatt tgtgtttgtc ttcatagtta aatatgcatt12601 atgatatcag cttgcataca ttttttaaat gacttgaata tctgacttta aaaattattc12661 tagaatttct gtgcttcaat attaatgcca gaagacttgg aattgtttat ttgtaggtaa12721 ctgcctttaa ggaaacttga ccaaatatta actaagttat gtatttcctt ttggcaacag12781

12841

12901

12961

13021 tgtgtgtgtg agagaaacag acagacaggc agactttttt ctatatgatg aaattaagtg13081 tattttaccc cagtaaattg caaggggtgg cagttgtgaa agcttctggc atgggaaagg13141 gatgtaacat ggtctttagc tggtttgttt tgtggaatgg aatttttatt tctgtccttt13201 gagtgactta cagcaatatt atacccttaa taagggtaaa ctaaactgtc cccccatctt13261 gaagggtcca agagaaagtt aatgtcatca ggatacatag cctatagata gcgacattct13321 ctagggaaag atggagatgc gcactacctg gccttcaaac tactcactaa tgaacacatc13381 tgagttgagt ttcacaccaa actcctggaa ccataacttt cttttcccag atctagtctt13441 gtttatcaca gacatcaaca gcctggcatg tttagcctca cttgggctag gtgcacccca13501 tcgtctcttg tacaagttct ctttctttct tttttttttt tttttttctg gagacagagt13561 ctcactctgt tgcctaggct ggagtgcagt ggcgcaatct cggcccactg caacctccgt13621 ctcctgggtt caagagtttc ctacctcagc ctcccgagta gcttgggatt ataggcacac13681 gccacgttgc ctggctatat atatatattt tttttttgag acggagtttt gctcttttgg13741 cccaggctgg agtgcaatgg cgcaatctca gctcactgca accgccacct cccgggttca13801 ggtgattctc cttcctcagc ctctaaagta gctgggatta caggtgcaca ccaccaagcc13861 cagctaattt tttatttcta gtagagatgg ggtttcacca tgttggccaa gctggtcttg13921 aactgctgac ctccagtaat ccacccacct ccccctacca aagtgctggg attataggcg13981 tgagccactg tgcccagccg cccagctaat ttttgtattt ttagtagaga cggggtttca14041 ccatgttggc caggctggtc tccaacttct gacctcaggt gatctgccca tttcggcctc14101 ccaagagtct ccagtctagt acgttgtcgt actcggtgtt gtaaaatcca aacaagggtc14161 agtttcccag gtaactggga aattcccaga atcacactct ttcgtcatag tgctcatcct14221 acaaaaaagg attgggggca ttttgtctaa aattaaatgt aaatggtgat ctgacataca14281 ggtggaaaga gaattgggaa gttttgttct ctcttctacc aacttgccac ataatcttgg14341 ccaagcaaag taacttgttt tttcttttaa tctttttaaa agaaatagag acacagtttt14401 gccatgttgc ccaagctggt ctcaaactcc tgcctgagct caagcagtct gcccacttcg14461 gcctcccaaa gtgctgagac tacaggcata agccaccatg cccctgggct cggccaactt14521 tttcgttttc ttttcaagag atgggggtct cactctgtca cccagcctgg agtatagtgt14581 tgggatcata gctcactgga gccttgaact cctgggctca agtgattccc ccctgtttta14641 gcctcctcag taaccgggac tagaggtgtc tgccaccaca cctggctaat ttttatatag14701 tttttttttt tttttttttt ttttaaagag atgacggtct tgctatgttg cccccagggt14761 ggtcttgaat tcttggcctc cagtgatcct tctgcatcag gctcccaagt agttgggtga14821 tctggctaaa gtaacttatt ttctgatact gtttacttat atttagaatg aatctcattg14881 gggttgcact ggggccgggc atggtggctc acacctgtaa tcccagcgct ttggaaggcc14941 aaggcaggtg gatcacctga ggtcaggagt tccagactag cctggcaaac atggtgaaat15001 cccgtctcta ctaaaaatac aaaaattagc tgggcatggt ggcacatgcc tgtaatccca15061 gctacttggg aggctgaggc aagagaatcg cttgaatcta ggaggcggag gttgcagtga15121 gtcaagatca tgccaccgca ctccaacctg ggtgacagag cgagactgtc tcaaaaaaaa15181 aaaaaaaaaa aaaaaaaaaa aggctgggca cggtggctcg cgcctgtaat cccaacactt15241 tgggaggccc aggcgggtgg atcacgaggt caggcgttcg agaccagcct gaccaagatg15301 gtgaaacact gtctctacta aaaatacaaa aataagctga aatcccagct actcgtgaag15361 ctgaggcaga gaattgctta aacctggtag gcggaggttg cagtgagccg agatcgcgcc15421 actgcactcc agcctgggga acggagtgag acttcatctc aaaaataaat aaataaataa15481 ataaataaaa taaaataata aataaagtaa aaagatctct cattgaacca gatgatatat15541 gaagtctctt ttaggaccaa tttcgagatt taaaaaattt ggcagaatta cttttttttt15601 ttgcagcgga gtccagcttt atcacccagg ctggagtgga atggcacaat ctcagctcac15661 tgcaacctct gcctcctggg ttcaagcgat tctcctgcct ctgcctccca agtagctgtg15721 attataggcg cccaccacca ggcccagctg atttttgtat ttttcagtag agttgaggtt15781 tcaccacgtt gtccaggctg gtctcaaact cctgacctta agtgatccgt ccaccttggc15841 ctcccaaagt gctgggatta ggtgtgagcc actgggctgg cccagaatga tttttaaaaa15901 gagatcagta aggccaggca gtggtggctc acgcctgtaa tcccagcact ttgggagact15961 aaggtaggtg gatcacctga ggtcaggagt tgcagacaag cctggccaac atggtgaaac16021 cctgtctcta ctaaaaatac aaaaattagc caggcatggt gacacatgcc tgtaatctca16081 gctactcagg agggtgaggc agaattgctt gaacccggga gtcagtttct tttttctttt16141 tttgagatgg agacccactt tgtcacccag gctggagtgc aatggtgcag tcttggctca16201 ctgcaatctc tgtctccggg gttcaagtga tcctcctgcc tcagtctcct tagtagctga16261 gactacaggt gtgcaccacc acacctggct aatttttgta tttttaggag agatggatgt16321 caccatgttg gccaggctga tctttaaact cgtgacctga agtgatccac ccgccttggc16381 ctcccaaaat gctgggatta caggtgtgag ccaccacgcc cagccctaaa gttgtatttt16441 gatggaacga actgttttga gaaataaatt ttaacgcgtt gagtctgaac tgggctgccc16501 tttcaaaatg tgaaggcccc ttaaagtagc acattggttg gttattcttt tatttattta16561 gatatatctg atctagttgt ctttgggaca aactcatatt taatatcata gctgcatgta16621 actgacagtg tagtctttgt cttcctgaag tgtttgtttg ttttttgaga tggagtcttg16681 ctctgtcgcc caggctgaag tgcagtggtg cgatcttggc tcactgcaac ctctgcctcc16741 cgggttcaag tgattctcct tcctcagcct cccgagtagc taggactaca ggcatgtgcc16801 accacaccca gctaattttt gtatttttag tagagatggg gtttcaccat attggtcagg16861 ttggtcttga actcctgacc tcgtgatctg cctgcttctg cctcccagag tgctgggatt16921 acaggtgcga gccattgtgc ccagctagta agtttttaag aaagattctc aaacctcttt16981 taaatcgtct gcctcacttg aagaggtatg ccctacctgt ttagggctgt agacccaggt17041 cattagaaga cagactaagt agtcctgggt gaacccatag ggcaccttca aggaggtaaa17101 attggtgatt ttagtttcac cagtagtttt tccctgaata tttattcctt ttgtgcttta17161 ttgatctatc tatatcaata aaaagtaatg gggcataaca aattatactt gtcattcttg17221 ttcattaggg caaatgttgt aggttgagtc aagtgtccag ccaacaagtt attttatgtg17281 tgtgtgtgtg tgtgtgtgtg tgtatacata tatacatttt tttttttttt tttcatcgag17341 acagggtctt gcactgtcgc ccaggctgga gtgaagtggt gcaatctcgg ctcactgcaa17401 cctctgcctc ccaggtttaa gtgatcttcc cacctcagcc tcccaagtag ctgggactac17461 aggcgcacac caccaccctt ggctaatttt tgtatttttt ttttggtaga gatggggttt17521 caccacattg cccaggctgg tcttgaattc ctgacctcaa gtagtccgcc cacctaagcc17581 tcccaaaatg ctgggattac aggcgtgagc caccacacct ggcatatata tattttaaga17641 tagagatggg gtttgctatg ttgcccaggc tggtcttgaa ctgctgggat tacaggcgtg17701

17761

17821

17881

17941 tgttatttat gttagggtga tgggggaaga aagggggagg gtgtattaac aagatacctt18001 gttttatata tgtgtgtgta tatgtattat tttattatac atacatgcat acttctgtag18061 ttccctggac tgtaggataa gttaggttac ttagaatctc aacagctagc atcgttttta18121 cttaggtttt caagcctact ggcagggtaa gcaagaggta gtaccatttt ggtaagaagt18181 agagagctag ggacagtaaa gatggagtaa tatatatatg agggtatagt caggccctag18241 aaattaatta tccagtttta tgctttttat aaaaaaactg agatggggtc ttgctatgtt18301 gcccaggctg gtctcaaact cctgagttca agggatctgc ccacctgggc ctcccaaagt18361 gttgggatta caggcatgag ccacagcacc cagccccagc tttatgcttt taattctaaa18421 actttttttg ttgtattttg cattcataag aatagatgtt aaataaacct tgaaatacaa18481 ccttggctca aacgttaatg gtcatggata aagtgaatta aaacttgtta ggggccaggt18541 gtggtggtta atgcctataa tcccagcact ttaggaagct gaggcagttg gatgtcctga18601 ggacaggagt tcaagaccag cctggccaac acagtgaaac cctgtttcta taaaaaatac18661 aaaaattagc tgggcgtggt ggcacacacc tgtagtccca accacttggg aggctgaggc18721 atgagaattg cttgaacttg ggaggcagag ggacttggga ggcagagggt gtagtgagcc18781 aagatcgcac cactgcattc cagccagggt gacagagcaa gaagactgtc aacaacaaca18841 aaaaatgtta tagaagtgaa aaaaattgat taatttagaa caagcttgtc cagtctgtgg18901 cccaggatgg cgtttaaatc agcccaacac aaatttgtaa actttcttaa aacattttgt18961 gatttgttgt tgttgtttag ctcatcagct atcattagca ttagtgtatt ttatgtgtgg19021 cctaagacaa ttcttccagt gtggcccagg gaagctgaaa gatcattatc ctctgatcta19081 tcatattaat gagctgcatc ctaaaagaca ttcatctata actaagctca gtttcatgtt19141 ttgttccttt ttcaatagat aagataggga atgagcaagt taataaagtg ggtattttaa19201 ttttaaggtt gaaactaagg atcataacat tatcagaggt ctagaactgg atggcagcta19261 cagagatcat ttagcctaat actggtttaa caaataatcc gggagatccg tgatatgtga19321 atgtgctagg cctgagatga gacagccaat tgtggaagag caaacactag aaccagtata19381 agttgcttac tgctttctta tgctattaat gagcatatcg cctcctgata tttatgatat19441 atggtcatgc caacagcttt gtcataaata gaactcccat ggcagcaatc acttaatctt19501 gtagttagag gtggggtctc accatgttgc cgagctggcc ttgaacttct gggctcagcg19561 attttctcca caggcacctg ctactgtgct cggtgcagca ctttgtgttt ttgaacataa19621 cctcaagatg ttattgtctt catagtaaaa caaaagatga ggcttagaac tggatcactt19681 tgcctgtctc ttcttacctc ctcccagttc aaaatgcttg catctcttaa tagctagcat19741 tcccttggat tttgcacatg agctcaaact caagcctcag cacaatcttt tttatagttt19801 tagtctttta gccagagtcg acttaccccc catacccact ctgcttcctt cataatgctg19861 ctttccctgg gcagagaatc cttgcccttc ttgtattatg tcactttgtg gggttggtgt19921 ctgctacact tacagcaagt ccagagattt tttttccacc acgtttgcag gagaactatt19981 ggcatggaaa atgacaattg ttttaatgtc aagtgaaact gaagttgatg ttcattgaga20041 ggtttctaat ttctagaggt gggttctttt tttggcatat gaagttgcag catattaaga20101 gaatttacag tagtacagat ggggttatcc catccacaac ttatgatggg gttacataaa20161 ctaaaaacgt gtttaataca cctaccccac cgaatattgt agcttgggcg tagcctaacc20221 tatctcagac gtgctcagaa cacttaaatg ttagcctaaa gttgggcaag atcatctaac20281 acaaagccta ttttataata aggaattgcc tatctcatgt aattcatcga atactgtact20341 aaaaatgaaa aacagtggct gcacgggtac cattataaag tcaaaaaatc ataagttgaa20401 ctgtcttaca ttatggtttt ccaaattttg atttgttttt aaatactctt tccttgcctg20461

20521

20581

20641

20701

20761

20821

20881

20941

21001

21061

21121

21181

21241

21301

21361

21421

21481

21541

21601

21661

21721

21781

21841

21901

21961

22021

22081

22141

22201

22261

22321

22381

22441

22501

22561

22621

22681

22741

22801

22861

22921

22981

23041 ttgataagta gtgctgtttg ccagctgtat attatcccta aaaataagta ataaggtata23101 tatggtacat attttgacat gcatatacat atttgcatcc tgactaggct gcccacagca23161 atttaagtta cttgaaactc gcttttatct tagtagccct ttggcctttc ttcagttttt23221 tttttttttt tttttttttt gagacatggt cttgctctgt tgcccaggct agaatatggt23281 gacacaacca tggctactgc agcctcgacc tcccaggctt aagtgatctt tccacctcag23341 cctcccaagt agctgagatt acagatatgc accaccatgc atggctaata tttaaatgtt23401 tgtagagaga tggggtctca ctgtgttgcc aggactggtc ttgaactcct gggctcaagt23461 gatcctcctg cctcggcttc ccaaagtgct gaggttacag gcatgaccca ttgcgcctgg23521 ccctttcttc agtctttaat aatcgaacaa aaggtttttg tttttagaca gtgtcttgct23581 ctgttaccca ggacagacct ctcgtgtcag cctcttaggt agctaggatt tacaggtaag23641 caccggcgtg ccctgcttta tttttttggt gggggaaggg ggaagggagt tgaagcttcc23701 ctatgttgcc caggctggtc ttgaactcct ggcctcaagt gatcctccag tctcccaaaa23761 gtgctgggat tacaggcatg agccaccgct cccggcccaa aagattttta aatgtgttat23821 acttcatgag acaggcttta ttttagatcg aattttattt atcaataaaa agttgagctt23881 tttattattt ggtgaatact gtttcaaggt gctttgttac actatctgtt gatccaacat23941 ttaaaaattg ttttattaca acctttgcat ttcagtgaat ccatctgcat acaattttaa24001 aagaatcatt ccttttttct gtagccaaat tgtcaaagat tctttcctac aattgatttt24061 tcaaagccct gagttaggaa tttacaattt ggcaaccatc tcaacttcat aagcaatttt24121 gttctttaaa tgtcacggcc aacattacct ggaaccattg ctgttttata gtttaggttt24181 atgttgtata ttttttttaa ttttttagag acggggtctt gctgttttca gactggagta24241 caatgggatg actagctcac tgcagcctca aactgctggg ttcaagtgat tctccttcct24301 cagcctcctg agtagctggg actacaggtg gtcaccatca cacctggcta atttttgtat24361 ttttggtaga gacagggttt ttcccgtgtt ggccaggctg ttcttgaatt cctgacctca24421 aagcgatctg cccgccttga tctccgaaag agctgggatt acacgcatga gccactgcgc24481 ccagccctgt tttttttttt tttttttttt taaataatgg tagtttactt gaatttgtaa24541 cacagtaaca caaaactatt ttgatctgaa cgcaagtatc taatggaaca gaataatata24601 cttcctttta gtgtgctgca tttggttact gggtaattta aaattcttcc tcagcacagg24661 tgttcaaaaa ccagtcttca gagattgttt tcatatcagt gtgccaactt tggcacattc24721 tgctaagtaa gaggcttaag tgtagcatgt ttctgctgtt ttgtgtttgt tttgttttgt24781 tttttgagac agagtctctc tgtcgcccag gctggagtgc attggtgcga tcttggctca24841 ttgcaacctc tgcctcccag gttcaagtga ttctcctgcc tcagcctcct gcgtagctgg24901 gattacaggc atatgccacg tgtattaggc actgctaatt tctgtatttt tagtagagac24961 gaggtttcac catgttggtc aggctggtcc tgaactgctg acctcgtgaa ctctgcccgc25021 ctaggcctcc tgaagtgctg ggattacagg cgtgagccac cgtgcctggc ctctgctcta25081 tcttttagct ttcccttggc acttctatgg tccagatgtt agagggtaag tattttgatg25141 ggggagatcg ttggactgta attgaaagtt atgtcttata atgaaatgtg ttatataaag25201 aagacctata aaacacttag gctgataaaa cccccaaacg atgaagcctc acttttaccc25261

25321

25381

25441

25501

25561 gttatcagaa agtcatttgt gacattagga ataacatact taggtgatca ttttccaaac25621 acagttacat aaaagtcagc cagtgactta ataggaagca aagggaaatt actccctgtg25681 ttataaaatt gagaattata tttagctgaa acatcgatgc ttaatgttaa ggggaatata25741 tgttaaaaag gggaaggagg tcagtcattc aggtcatgag gccctttgac ttgaattcat25801 ttcctcagaa ggtaggtata ttcatagtga acaaaaatac aaaggctgta tgaaaagatg25861 aaaatgttac aggtttatcc ttaaattaga ctcatttgca gaaatgcaaa tgaggtaaga25921 aagcaaatat agttcatgac ctctagcaac tgttgaaaac tgctctttag ggatgacatg25981 ctggcccttt tttttttgtt gttgccaagg ctgaagtgca gtggcaccat cacagctcac26041 tgcagcctcg aactcccagg ttcaaccctt cctcctgcct cagcctcccc agtagctggg26101 actacagatg tacaccatca tgcctagctc atttttaaaa aaatttttta tggcattgta26161 tttatcttct ctttataacc aggggttgac cagccacaga acttgtaaag ttttttatat26221 ttttaaaagg ttgtaagaaa tagtagtagt tggctggtcc ccgctgctct cctgcattat26281 agtatacttc tgttcaccta gtttgctaga gagaggcagt atagtgtgta tagtgatttc26341 caaacttttt ctttaaatca gaatcacctg aaagaatgtg acaacgtgta aaaaaaaaaa26401 aaaaggtgca gagattccat cctgacatgg attcacttga ttggaattga ttctaggcat26461 ctcagtagtt ttaaagagct cctaggtgat tctattctgg ccagcgttga gaatcactag26521 ggtagtgggt tggtaagcag gctctgatgt tttaaaggcc aggtgaggcc ctatgcctct26581 tgtctctctt agcctcaact ttctccatgt tagcaaatgg atttcagaac agaaccaacg26641 tacatgtgat tgtgaaagtt gttttagagt gcctagctct tacgtaaggg ttcataagaa26701 agacaaaagt ttatgaaact gttactacca gtcataaaag accttttcct ccctcattca26761

26821

26881

26941 tttgcttctt gtatatgagc cttttaaatc taatatttga tttttctggt gttactttaa27001 aaacatcact ttttaagaac tgcatagtct ctctctcttt tttttttttt tgagatggag27061 tttccctctt gttgcccaag ctggagtgca atggcacgat cttggctcac tgcaacctct27121 gcttccaggt tcaagtgatt ctcctgcctc agcctctcga gtagctggga ttacaggcgc27181 atgccatcac gcccagctaa ttttttgtat ttttagtaga agcggggttt caccatgtta27241 ggctggtctc ttaactcctg acctcaggtg atctgcttgc ctcggcctcc caaagtgctg27301 ggattacagg cgtgagccac cgtgcccggc caataattgc atagtctctt aatgagattt27361 aatcttttat accaatatgt gtagctcatg atagctatat aacctagaag atgaatttat27421

27481

27541

27601 aaaaacattt gtacaaataa ctatttttat agaagattat ctgaagtaca tttaaacaat27661 atgaatgttt ttagagcacg cactcaccat tgtggcacag accgatagtt ggagataaaa27721 ggtgatattg tgaaaggttt ttgattaccc attaattatt aggccttaca ctgtttagtt27781 gtaataaaac atttgttata ctacggggat gagaacacta ataggaggac tcaggaagtt27841 tatgaccttg agcgatactg tattttcttt aaaagaaacc tcactcccca tgggctgcta27901 agcagactcg tgtagctaaa caaggcctat ttatagaatg cttttagacg tggatgtact27961 aaccgatgtt gcttttctgt cctagcattt ttgttttaat tccttttttg ttttaattcc28021

28081

28141

28201

28261

28321

28381

28441

28501

28561 cagtaaaagg ggaagggatg atgcactatg aaaaaacaaa aaaacttttt tttttttttt28621

28681

28741

28801

The genomic sequence for human MLH1 (found on human chromosome 3) hasGenBank Accession No. NG_007109.2. Sequence information related to humanMLH1 (isoform 1) is accessible in public databases by GenBank Accessionnumbers NP_000240.1 (protein) and NM_000249.3 (nucleic acid). Sequenceinformation related to human MLH1 (isoform 2) is accessible in publicdatabases by GenBank Accession numbers NP_001161089.1 (protein) andNM_001167617.1 (nucleic acid). Sequence information related to humanMLH1 (isoform 3) is accessible in public databases by GenBank Accessionnumbers NP_001161090.1 (protein) and NM_001167618.1 (nucleic acid).Sequence information related to human MLH1 (isoform 4) is accessible inpublic databases by GenBank Accession numbers NP_001245200.1 (protein)and NM_001258271.1 (nucleic acid). The genomic sequence for human MLH1(GenBank Accession No. NG_007109.2; 79,540 bp in length), is found atnucleotide no. 5,001 and terminates at nucleotide no. 62,497, whereinexon 1 is located between nucleotides 5,001 and 5,314; exon 2 is locatedbetween nucleotides 8,270 and 8,360; exon 3 is located betweennucleotides 12,606 and 12,704; exon 4 is located between nucleotides16,052 and 16,125; exon 5 is located between nucleotides 18,642 and18,714; exon 6 is located between nucleotides 20,465 and 20,556; exon 7is located between nucleotides 23,471 and 23,513; exon 8 is locatedbetween nucleotides 23,662 and 23,750; exon 9 is located betweennucleotides 26,083 and 26,195; exon 10 is located between nucleotides29,157 and 29,250; exon 11 is located between nucleotides 31,961 and32,114; exon 12 is located between nucleotides 37,288 and 37,658; exon13 is located between nucleotides 40,435 and 40,583; exon 14 is locatedbetween nucleotides 51,837 and 51,945; exon 15 is located betweennucleotides 53,919 and 53,982; exon 16 is located between nucleotides59,170 and 59,334; exon 17 is located between nucleotides 60,168 and60,260; exon 18 is located between nucleotides 60,555 and 60,668; andexon 19 is located between nucleotides 62,137 and 62,497. It isunderstood that intron sequence precedes and follows the denotednucleotide regions comprising the exon sequences listed herein.Oligonucleotide compounds (e.g., exon skipping or intron retaining SSOs)can be directed to the nucleic acid sequence corresponding to the regionof interest for each of the exons 1-19 described herein, and intron-exonjunctions, or exon-intron junctions listed with GenBank Accession No.NG_007109.2.

The genomic sequence for human PMS1 (found on human chromosome 2) hasGenBank Accession No. NG_008648.1. Sequence information related to humanPMS1 (isoform a) is accessible in public databases by GenBank Accessionnumbers NP_000525.1 (protein) and NM_000534.4 (nucleic acid). Sequenceinformation related to human PMS1 (isoform b) is accessible in publicdatabases by GenBank Accession numbers NP_001121615.1 (protein) andNM_001128143.1 (nucleic acid). Sequence information related to humanPMS1 (isoform c) is accessible in public databases by GenBank Accessionnumbers NP_001121616.1 (protein) and NM_001128144.1 (nucleic acid).Sequence information related to human PMS1 (isoform d) is accessible inpublic databases by GenBank Accession numbers NP_001276337.1 (protein)and NM_001289408.1 (nucleic acid). The genomic sequence for human PMS1(GenBank Accession No. NG_008648.1; 100,545 bp in length), is found atnucleotide no. 5,001 and terminates at nucleotide no. 98,545, whereinexon 1 is located between nucleotides 5,001 and 5,509; exon 2 is locatedbetween nucleotides 12,706 and 12,857; exon 3 is located betweennucleotides 16,685 and 16,867; exon 4 is located between nucleotides26,568 and 26,670; exon 5 is located between nucleotides 38,933 and39,096; exon 6 is located between nucleotides 64,880 and 64,996; exon 7is located between nucleotides 73,571 and 73,693; exon 8 is locatedbetween nucleotides 74,855 and 74,998; exon 9 is located betweennucleotides 75,155 and 76,044; exon 10 is located between nucleotides84,659 and 85,144; exon 11 is located between nucleotides 88,715 and88,845; exon 12 is located between nucleotides 94,412 and 94,572; andexon 13 is located between nucleotides 98,188 and 98,545. It isunderstood that intron sequence precedes and follows the denotednucleotide regions comprising the exon sequences listed herein.Oligonucleotide compounds (e.g., exon skipping or intron retaining SSOs)can be directed to the nucleic acid sequence corresponding to the regionof interest for each of the exons 1-13 described herein, and intron-exonjunctions, or exon-intron junctions listed with GenBank Accession No.NG_008648.1.

The genomic sequence for human PMS2 (found on human chromosome 7) hasGenBank Accession No. NG_008466.1. Sequence information related to humanPMS2 (isoform a) is accessible in public databases by GenBank Accessionnumbers NP_000526.1 (protein) and NM_000535.5 (nucleic acid). Thegenomic sequence for human PMS2 (GenBank Accession No. NG_008466.1;42,868 bp in length), is found at nucleotide no. 5,001 and terminates atnucleotide no. 40,868, wherein exon 1 is located between nucleotides5,001 and 5,110; exon 2 is located between nucleotides 8,076 and 8,215;exon 3 is located between nucleotides 10,049 and 10,135; exon 4 islocated between nucleotides 10,315 and 10,417; exon 5 is located betweennucleotides 11,471 and 11,654; exon 6 is located between nucleotides14,832 and 14,999; exon 7 is located between nucleotides 16,684 and16,781; exon 8 is located between nucleotides 18,474 and 18,573; exon 9is located between nucleotides 22,050 and 22,134; exon 10 is locatedbetween nucleotides 24,152 and 24,307; exon 11 is located betweennucleotides 26,487 and 27,348; exon 12 is located between nucleotides31,116 and 31,283; exon 13 is located between nucleotides 35,411 and35,511; exon 14 is located between nucleotides 36,350 and 36,519; andexon 15 is located between nucleotides 40,565 and 40,868. It isunderstood that intron sequence precedes and follows the denotednucleotide regions comprising the exon sequences listed herein.Oligonucleotide compounds (e.g., exon skipping or intron retaining SSOs)can be directed to the nucleic acid sequence corresponding to the regionof interest for each of the exons 1-15 described herein, and intron-exonjunctions, or exon-intron junctions listed with GenBank Accession No.NG_008466.1.

Sequence information related to human MLH3 (isoform 1) is accessible inpublic databases by GenBank Accession numbers NP_001035197.1 (protein)and NM_001040108.1 (nucleic acid). Sequence information related to humanMLH3 (isoform 2) is accessible in public databases by GenBank Accessionnumbers NP_055196.2 (protein) and NM_014381.2 (nucleic acid). Thegenomic sequence for human MLH3 (GenBank Accession No. NG_008649.1;44,769 bp in length), is found at nucleotide no. 5,001 and terminates atnucleotide no. 42,769, wherein exon 1 is located between nucleotides5,001 and 5,153; exon 2 is located between nucleotides 6,815 and 10,157;exon 3 is located between nucleotides 14,056 and 14,154; exon 4 islocated between nucleotides 14,833 and 14,918; exon 5 is located betweennucleotides 16,518 and 16,622; exon 6 is located between nucleotides18,121 and 18,193; exon 7 is located between nucleotides 23,043 and23,114; exon 8 is located between nucleotides 24,354 and 24,465; exon 9is located between nucleotides 25,831 and 25,990; exon 10 is locatedbetween nucleotides 33,515 and 33,538; exon 11 is located betweennucleotides 33,641 and 33,719; exon 12 is located between nucleotides37,553 and 37,704; and exon 13 is located between nucleotides 39,332 and42,769. It is understood that intron sequence precedes and follows thedenoted nucleotide regions comprising the exon sequences listed herein.Oligonucleotide compounds (e.g., exon skipping or intron retaining SSOs)can be directed to the nucleic acid sequence corresponding to the regionof interest for each of the exons 1-13 described herein, and intron-exonjunctions, or exon-intron junctions listed with GenBank Accession No.NG_008649.1 and described in SEQ ID NO: 1. The human MLH3 gene sequence(GenBank Accession No. NG_008649.1; found on chromosome 14) is depictedin SEQ ID NO: 1 below, where the bolded italicized nucleotide basescorrespond to EXON regions.

SEQ ID NO: 1    1 gatcatttga gcctgggagg ttaaggctgc aataagctgt gactgtgcca ccatccttca   61 gaaaaaaaaa agaaaaagga aaagaggtat tgacaattca cattcatgtt tcaaagattc  121 cttccaggtt agaatttgaa ttttaagtac cacagtccca ggaatgagac acttattttt  181 catttttatt ttttagcttt agttttagta tgaggataat gctggcttaa taaaatgtgt  241 tgggaagtgt ttcctcttct tttttttaac ccttaattct tggtttggat cttcttctat  301 tttttggaag agtttgtgaa gggttggtaa aggatttttt tttaaacatt tggtagaatt  361 tacccatgaa gccatctggt cctgggcttt ttatctgtgg gaagtttttg attacaaatt  421 ccatctcttg gtataggtct attcagactt tctatttctt cttgattcag ttttggttgt  481 ttgtctttct aggtattagg tttgttttca tccaaaaaaa tttaatctaa ttttttggca  541 tataattgtt catagaattc ccttttatcc tttttatctt tgtaaggttg gtggtaatgt  601 ctccttttat ttctgatttt agtaatttga ggctttcatc cgtttttctt tatcagtcta  661 gctaaaggtt gtcaattttg ttgatcctgt caaagaagca acttttgttt tattgatttt  721 ctctattctt ctgctttcca ttaatttctg ctctaatctt tatttccttc tttctgctca  781 ttttgaattc agtttgctct tctttttcta gtatcctaag gtggaaatct tgattattga  841 tttgaagaga gtttcttttt agattgcaac tagaggtggt gattacagtc cttgaaatgt  901 tgtcagatga ttgtttaagc aaaaatatgg ttatgaggta gaattcactt tttggaattt  961 tcttagggga gaaaaaccct gttggtgcaa tagatattca aaactgaaca tgagtcaata 1021 acattatgtc atcattaaaa aaataagaac agagttgagc acacaaatga aaccagtagg 1081 aaacgcccat tctcaacact ggtcaaattt tggagtgttc tgttcaactc tcaagagttc 1141 agataaggcc aggtgcagtg gctcatgcct gtaatcccag cactttggga ggccgaggca 1201 ggcggatcat gaggtcagga gatcgagacc atcctggcta acacagtgaa accccatctc 1261 tactaaaaat acaaaaaaat tagccgggag tggttgcggg cgcctgtggt cccagctact 1321 ggggaggctg aggcaggaga atggcataaa cctgggaggc ggagcttgca gtgagctgag 1381 atcacgccac tgcactccag tctgggcgac agagcgagac tccgtctcaa aaaaaaaaaa 1441 aaaaaaaaaa aaagagttca gataaatcac attgcaaatt tttaaagatt tatttgactg 1501 tgacaacctc ctgtttaaaa ctgcacattc ttacagctat ttaccatata agataactct 1561 taagaactgg agatagtcag ctcccctggg ttaatttgaa gcagaagagg gcagttgtta 1621 tactgccctg tcagttggat gcggagtctt actcaaaatt cattctcagc attcttcttt 1681 tatggtatct tctttggcac ttagcagcgc atcaggtagg catcttctat ttttcttcat 1741 tccttaattt cctttgtatc cctcaaatgg ttatttattt ggctggagtc tgttttgttc 1801 attaagcaaa catgtctttg ctttgaacat gtctttgatt tgatggatac ttaaattcct 1861 catcaaacat tttgttgcta tgcataacgt tttctttggc caactccagc aatttcccac 1921 attttgacat gcaatcatgt taactcccat tttcttttgt aatccaacat cttctattta 1981 gataattact ttaacaatca atgacttaat attctaatca taaatttata caaaaataaa 2041 attacctcca aaacattgct acctttccta aacattcagt cttgccacag tttaataaaa 2101 ggaaagaaca ttaaaaagga taagacactg taatgattag atgcttttta taagcctaaa 2161 ggcattgtga ttatttagac agaagagaag aaagtgaagt gaaaacctga tagttatgta 2221 gtctcatggt ttgctgttga gaggctgaac accagctgct ttccttttct aggaagataa 2281 taaagtgggc tttggctaca acataaagat gttgggttag acagtttcac tacagtaaga 2341 acaacgggat gagttgccca ggaaattgtg aaatactttc taatgatctt taaagatata 2401 atgaacacta attcatctgg atttgtttac gtgtggtcct ggttaaaggc aaagggaagg 2461 atcagataac ttcatgtttt ttccatttaa catacccaat agattcttga ttaggggaag 2521 ggaaaatgag caagatacag tccagtattc taaaaacaat cagccttagg ggatcatttc 2581 aaaagcatct gttttggact taagtctttg atacttaacc aaattgacta cacagtgaaa 2641 aattctagtg cctgggtttt atagggtaga agaaagacat gcagtcaagt ggccaatact 2701 tcatgtgaag ataagcaatg agatccttct tgctgtcttt cttttgactg ttctgggcaa 2761 tatcaaatta gtttcagtgg cttgattcta ggccaagatt ctggcaacag attgtagtct 2821 taccttgttt tcttcaatct cactggatct ctctctcttt ttacccccct taggctgagg 2881 gtaaaaagct gggattggta ggctgggtcc agaacactga ccggggcaca gtgcaaggac 2941 aattgcaagg tcccatctcc aaggtgcgtc atatgcagga atggcttgaa acaagaggaa 3001 gtcctaaatc acacatcgac aaagcaaact tcaacaatga aaaagtcatc ttgaagttgg 3061 attactcaga cttccaaatt gtaaaataat ggcctgaatt taagttttct aagataaact 3121 cagtggtttg gtttttatta ttaatagaga tagaactatt gtgtgttaat attagcatta 3181 gtcaataagt tattttaatg tcagattttt gaatgttatt atatattacc tgtatgatgg 3241 aaggattacc actgtacaca aatctaatca ataaaaacgt tagaaccttc tgcttagagt 3301 acttttaaaa aatcttcagt gaacttcctt ttgggcgaaa tgagaggtct ttattcagta 3361 aacatttgta ggaagaggat tttgaggtaa tttaaagagg tctgaaagaa aaaaagtctg 3421 agtcatttct ttaaatggtt tctatgaaat gttcttcaag aaattccatg cctaataaga 3481 acaaatacca caagttcaat ttgttagctc tgttcacctt atgtttggat gaattacttc 3541 tgctgttgtt tcttttctct gggtaaggaa ttcacataaa gttatgttat gggctgaact 3601 gtgtcccacc aaattcatat gttaaagtct cagtccccag tacctcagaa agtgactgta 3661 tttggacata gggcctttaa agaggtgatt aagattaaat gaggctgtga gggtgggccc 3721 taatccaatc tgactggtgt tcttataaga gaacatattg gctatagaca cgtgtgcaaa 3781 aatcaaagac cctgtgaaaa tggccatcta caagccaagg agagaggcct caggagaaat 3841 cgttgctgcc aacaccttga tctcagactt ccagtctcta gaactgagag gaaatagact 3901 tctgctgctt aagctactca gtctgaggta ttttgttatg gcagccctag gtatagtaat 3961 aatcataaac agttatcagg attttgctta atcagcccta gaagactggt tggttgggtt 4021 tggttagtca ctaaactagc atatatcaaa tgcttaccag gtctgacaaa ttcgttataa 4081 attccacttt aaattctcaa tgaaaatgag atagaaagca aaaactaagg actggttaac 4141 aattccaaaa cactttattc tcacagcatt ctcagagttc tgctctcctt tagttatttt 4201 tataactaaa agctgtggtg gcactgggga gattcaagtc agtgaagaga gtcttggtgt 4261 tgtcatctgt aaattaagag ttgagcaaag gccgggtgcg gtggctcacg cctgtaatcc 4321 caacactttg ggaggccgag gcgggcagat cacctgaggt ccggagttcg agaccagcct 4381 gaccaacatg gagaaacccc cgtctctact aaaaatacaa aattagccga gtgtggtggc 4441 gcatgcctgt aatcctagct actcgggagg ctgaggcagg agaatcactt gaaccctgga 4501 ggcggaggtt gcagtaagct gagatcgcac cactgaattc cagcctgggc aacaagagct 4561 aaactccgtc tcaaaaaaaa aaaaaaaaaa aaaaagagtt gagttagaca gtttctcagc 4621 cttttccagc tccaaatgcc ataattctaa gatggcaggc tctggaatta ttcattcatt 4681 cggtgcctac cataggccag gcactgttct tggtaactgg gatacagcag taaacaaaat 4741 atataaactc cttaccttca catagcttac attctaggga gagaagacaa taagtaaaca 4801 cataaaatat atattgaatt aaacggtagt taagagcaaa ggtgaaaatg agaaaaaaaa 4861 ggaaagggaa aacacgggaa aaaaaaataa aaacaaaagt aaaagctacg acatagtctt 4921 taacaccatg ccaaaaggga ataaggattg agactgtagg taccggttca ctgaaccctg

 5161 tgggggaagc acgcgacgaa aagatgatgc cggggtctct tctaacacca gaagggccct 5221 gatgatggct gcgcgcagct ttcggagccg gatgcgcgag ggccccggag gcccggcggc 5281 ctggcggccg ggcgggccca gtttggggac aaggacgggg ctggccaggg aggggctggg 5341 cctggcggga aggcagcgct gccccggact cggcccgcgc ggccctccca ggcccccgtg 5401 ccctggatcc aggcccgtgc gtcccgtcag tcccaggcgc tgggaggcgt catcaggaaa 5461 tcattgggtt tacattaatc ggaatacttg ttaaacgttt acccgtcagt tgctgccggc 5521 tgcttagtgc attagcttag aaagcagcag aaattctgca gttaagagcc ctgatttgtc 5581 ccgagtttgg aaaaccggcc ccaccgcctg caggggccca cccacgtggc tctcactgat 5641 ggagaagaag gggagacctt taatggcact ggaatcttag ggtttggttt tttttgtttt 5701 gttttttgtt tttgtgtttt cacttgcagg aaaacttaaa atcaagttca ggcagcccca 5761 tgccatcatt attattacca aggtagttta tgcccatctg taaagaccaa aagaatatta 5821 ataatgacct tctggccggg cgcggtggtt cacacctgta atcccagcac tttgggaggc 5881 caaggcggga ggatcacttg agttcaggag ttcgagacca ggctggttaa catggtgaaa 5941 ccccatctct acataagaga caaaaattag ccaggcatgg tggcgagcgc ctgtaatccc 6001 agctactcgg gaggctaagg ctggaggatt gcttgagccc gggaggtgca ggctgcagtg 6061 agccgagatt gtgtcattgc actccagcct gggtgacaga gtgagactgt ttcaaaaaag 6121 aataataacg actttctaaa aacatcagag taatacatga acaggattta acaatcaaat 6181 ggcaaagaaa ggtttatatg aaaaggaatt ttcctgcttt atcttttccc attcccagcc 6241 tctttccctg aggtgactat acttaaccat aataataata acctcttaat gcccccaatc 6301 tctctttgat acaatttgat tcttaaaatt tcttcctatt gcttcacctt attacaatgt 6361 cttagcaatt cattataagt atttttctaa aagtcatggt tattgtgtaa tgtataaatg 6421 ttatgaaact agcttggaca tttttgtcat gaataatttc tagctaatgt tccttagctg 6481 tatttaattt aggcatctgt ttttggtgaa gtggttagaa tttcgaatac tgtgtttact 6541 ggtcaacttc aagtgtaatt atgatttcac tttaggacat gtgggattta gaaaggagca 6601 ttgaaaatta tgaaattatg aatttttttt ggatgttaat ccattgcacc aagcatgagc 6661 tgtgcctaga gatcagcggt ataactttgt tttgctttgt ttcacaattt ggtttaataa 6721 gagtgatttc atttacctca agtgctattt cttcataatg ctgtgtaatg ctaaagcttt

10201 ggaatgctgg acaaggagta agatcctcat tatccaaaga gattatctca acagatagaa10261 cattttgaag accacttata aaacatatgt tgtattttca tgctatatga agatttgcta10321 cgtccaacac tacttttttt tttttttttt tttgagacgg agtcttgctc tgtcacccag10381 gctggagtgc agtggtgcga tctcagctca ctgcaacctc tgcccccctg gttcaagcga10441 ttctcctgcc tcagcctcca gtgtagcttg gattacaaga gcatgccacc atgcccagct10501 aaattttttt attttcagta gagacaggat ttcactatgt tggccaggct ggtctcaaac10561 tcctagcctc aagtgatcca cctgcctcgg cctcccagag tgttgggatt ataggtgtga10621 gcccgcacgc agcctctagg tccaacacta tttaaatgga gaagtacagt gaaaggatgt10681 cagtgtgtgt atatgtgtct atgggtgtgt gtatgtgtaa gaaagggggt gatgggtata10741 tacctagagc aacaacaaaa ggggtgagac tgtccaattt taataaaaat gatactatta10801 atagttggga agatccacag tactttccta ccttgttttc ccttcctgtt aaatgcccag10861 ctttcctaga aatgtgtctt taaaggcacc tgcagtgtca gcatggtatt ttactggaag10921 tttctctgcg tggcttccag gttaagggcc agaatgattc tgtaatcatc ctaggtttcc10981 cagtaaactc tatggcttag tacagtacta catttatgag ctttttcttt ctagaaagta11041 ggacttgtcc atgaattttc aaaatatagg tcaagtatat actttcatga ctatactttt11101 caaatgtact ttattataat gcatatggta aaatctcctt gatgtgttta tggtacaagt11161 agctgttatt caggaagaca ataatatggc atttatttat ttgagacaga gtttcactct11221 tgttgcccag gctggagtgt agtggcgtga tctcggctca ctataacctc cgcctcccag11281 gttcaaggga tcctcctgcc tcagcctccc aagtagctgg gattacaggc acccgctacc11341 acacccggct aattttttgt gtttttgtag agacagggtt ttaccgtgtt ggccaggctt11401 gtctcgatct cctgacctca ggtgatccac ctgcctcggc ctcccaaagt ggtgggatta11461 taggcgtgag ccactgcacc caaccaatat agcttttaga tcgtaccaca gggctacatt11521 agagcctgca ctaggactct gggttctcgg tccacattta aagtgatatt gctttggcag11581 tgtgttgaga gccagatgtt gggaggtgat aaagctgatt tccatctggc tataatcttg11641 gctttgcact aggaggataa acattacatc tgtatccgta tttaaaattt tgaatgctgc11701 agaagaatgg aacatgtata aatgtagtta taagtcaatt tctaatttct cctgacgatg11761 aatatctcta gcagaaagct gtggtttctt taaggttaaa tgaaaccttt acttttcagt11821 gtttttctgt ttttccttgt gaaaaaaaaa tcactgtagt aacttcagta gtatttcata11881 gtattttgta gtattcatta ggagaaaact catttctttc actgttgttc ctgagactat11941 caagttgctc ttaaagccac tttatgtgct tttcaatcta tgtggtttta tgtcctgact12001 tacactgaag gcttttcaga ggtaaatcta cagcactgac acctcatctt cctgaaatgc12061 aactgcttct gatatggtgc caaaaaataa ctggattaca ttatactgtg agatggctgt12121 tgctaaaaga aagggggaaa tcttagctgc catattcaac gtaaatattt cttccttttg12181 aatatagacg tttaaaacac tttaacctac ttaacattct ggattagcat acttttttat12241 gaacttttga gggtcatgtt aactgatagt ttcctaatga aaaatatttt tggtatagaa12301 agaagttcca tgaacagatg gctcctcagt accctgctac ctttctggaa atggctgata12361 tacatcatga catcaggaca ttgtcaaact ttacataaaa gtgttacatg aaataaattg12421 ttaaaagaca attttttttt tttgagacgg agtttcgctc ttgttgccca ggctaaagtg12481 aaatggtgcg atctcggctc actgcaacct ccgccttctg agttcaagcg attctcctgc12541 ctcagcctcc tgagtagctg gaattacagg catgcgccac catgcctggc taattttgta12601 tttttagtag agacgggatt tctccatgtt ggtcaggctg gtctcaaact cccaacctca12661 ggtgatctgc ccgcctcggc ctcccaaagt gctgggatta caggcgtgag ccaccagcct12721 aaaagataca ttttaatgta cagaaggagg atctataaag gtgattttga ggagtagtgt12781 caagtcccca agccttggta attttttctt taaaatatat tttatgcagt tctttctatt12841 gtagttataa accttatttc acttcattat cttaatttta tcattcttaa cctctccagg12901 tttagtccaa cagatttatc tctcatgttt tcctcctgtt ctaaagctgt caaattcagc12961 attgtttcta aaactcaaat ctgattatcc ttcatggaga agaacagcat ttgggccaga13021 tcttactaga ctctggagtt tgaattgaga acctttcatt tgtctacctg ttcacttttt13081 catgcagcat ttgttgatca tccacactgt gctaggtgct gtcacgttgc aggttaactg13141 actttgcttt taggagttca cagtactatt agggaagata gctatctaaa tataattgta13201 ttataatgag acaaatgtta ggatagagac atacattgga gataagagac ataagttgga13261 gatttgtgaa ggttgcacca agttatggct gacaagtact gctgtatgtc taacacaggg13321 cttctctggg taattttttg ttgtaaggtt tgtcctatgc attgtagaat atttagcagc13381 atccgtggcc tttatctgct agatgctagt agaacgtaga accctttact caagttgtga13441 caatgaaaag tgtctcaagc cgggcacagt ggctcacgcc tgtaatccca acactttggg13501 aggccgaggc gggtggatca caaggtcaag agtttgagac cagcccggcc aatatggtga13561 aaccccatgt ctactaaaaa tacaaaaaaa ttagccgggt gtggtgacgc atgcctgtaa13621 tcccagctac tcgggaggct gaggcaggag aattgcttga acccgggagg cggaagttgc13681 agtaagccga gatcacgcca ctgtactcca gcccgggcga cagagcgaga cgccgtctca13741 aaaaaaaaaa aaagggtctc aagacattgc caatgtcccc tcgtgcaaaa ttgcccccca13801 gtgagaatca cagtgaccta ttctaggtct tcattctgtc tggtatgaat agataatgga13861 actaagcatt aatatagagt cctatatgga cagcataatt tcattaaagg atttctgatt13921 ctcaatctag tgtttagtag tctgctttat gtcttgactc agtttgtgca gaaagaggtt13981 ttatgtgatt aaatttttaa agagtttact tgtattttaa gttcacattt ctaggttttt

14161 gtggcagaga gtgctggggc acacagtaca catcgtacca cctaactcaa aaaacaaagc14221 aggcttgcat taagcttgat cagtgccagc tgtgctgtac tggaatcagg aattccccac14281 ggccctgttt aacagcggaa ctaattaatt cagcgcatgc atttaggtca gaaattactt14341 ttccagactt gaggtcaata tttgctgatt atgcatagtc tgtggtgtct tgtgattagg14401 ggacatgttt cttattttta aaaaggctgg acttgcactc caagttgctg tagttttatg14461 taagcaaaac tcttcaaaaa aagctacaga catcaatttt gtgtgtatgt ttaaaaatag14521 ataaggtata ttttgcttaa aggcagtaag gctataaatt tgagccatag cttctctgtc14581 agtattccac aatatctctt ttaacagttt ggagaaaagc tgtaatgcca gaagatacag14641 tcaccaactc tagtttaaaa agtaacagat caaaataatt tagagataca gcaaacactg14701 aacatttgta gaacatagaa acaatgtacg tgacctttgg tcttgaggct atggctcacc14761 aaacaggaaa tgccattccc tcctttccct gtggttctgg atgccaactt tacctgttcc

14941 ttgccttttt tttttcttct tcttcttctt attttttaaa ttttgagatg gagttttgct15001 cctgttgccc agactgaaat gcaatggcat gatctcggct cacgtaacct tcgcttccca15061 ggttcaagca attctcctgc ctcagtctct cgagcagctg ggattacagg cacctgccac15121 cacgcccagc gaattttgta tttttagtag agatggggtt tctccatatt ggtcaggctg15181 gtctctaact cccaacctca ggtgatctgc ccgcctcagc ctcccaaagt cctgggatta15241 caggtgtgag ccatcacgcc cagcctttct ttgcctgtta ttttaaactt tgagtttcaa15301 gatgggtaca gtggcatgca cttatagtct cagctactca ggaggctgag gtgggaggat15361 cacttgaggc cagaagttca aggctatata gtgtgctgat tgcacctgtg actagccact15421 gcacactata gcctgggcaa cacagtgaga ctccatctct taaaaaataa gtaaattagt15481 taaaaataaa atttgaattt tatttaagct gggtatgggc atgcaagact atattaattt15541 gtaattatga ttggaaactg ggcatatttc caaattatgt atttggaaat tactgtttaa15601 tgtagagggg agaaataaaa tttatagcat ttgggaactc aggcattttg gattaaacat15661 gtataatcaa tgtgagaaca gtgaatgtgt ttaccactct cagaaaccct gacttgtagc15721 tttggcacag tattcagggt ggtgataatg aaattgttat cattagataa agccacacat15781 cttctactgg aggaacagtt cctcattggc cccgcatgga tgttctctcc tcagtgtatt15841 ttcacagaat gtactgtgca tgttataaac agggtgtata atagatgagc atttgtttac15901 cagttctttc ctcaagatat catttgagga cttgatcatg gaacatagac aaattgttgt15961 tgtgaaacaa ttttttttgt tttgtcagta attttgttta taagcaaaaa tttttatgta16021 agactcatta ttcataaatt cttttgaatt tttttttttt tttttttttt tttttttttt16081 tttgtgacag agtcttgctg tgtcgcccag gctgaagtgc agtggcgtga tctcagctca16141 ctgcaagctc cgcctcccgg gttcacacca ttctcctgcc tcagtctccc aagtagctgg16201 gactaaaggc gcctgccgcc acgcccagct aattttttgt attttttagt agagatgggg16261 tttcaccatg ttagccagga tggtctcgat ctcctgacct cgtgatctgc ccgcctcagc16321 ctcccaaagt gctgagatta caggtgtgag ccaccacgcc cggccgattc ttttgaattt16381 ctataaattc cctgaattaa acccacctct agtaggctaa ataaaaatga atgcttttaa16441 agcactctga tatctttggt aaaacaattc tgacacaaaa attaaaattt caattatatt

16681 atatatggcc taactcttct aaaatttgat ttatttaatg atatagacca ccttggtcaa16741 ggcacgtgtg tgtgtgtgtg tgtgtgtgtg tgttcttact agaactttta tcatcagaag16801 aaaaagcaag ggattaatta taattacaaa gaaagaaata tctttagtag tgtaagtagg16861 tttggaggga atgaattatg gattcaggct gacagtttca aggattctca aggttctcct16921 gcagaagatt atatattgac tttatatttg gcaataattt ataaatcaaa gtcctttagt16981 gaccttgtcc ttaagtgtgc aacagtgccc ttttagaaat taccttctat tgtgtgagcc17041 tacacaggtt tgggaaagcc catattgaga agtcatttca caaaagggac cctttgtctg17101 atctgctttt ctattttgcc ttgtgtttac caccatggct gtcttattcc ctttgttttc17161 agtacggaaa caaattagga ggctaacaaa tttgggtaaa ctcctaacct ggcattgacc17221 tggtaccaga gcaggcaagc agttgatcca gaaatagacc ctggggcagt ggtataggct17281 cgggggtatt ggagagcctc aagtacctag acagagagga accaccaagg tctaaagcca17341 ccagtggctt ttaacttact tctttaatag actagcagcc gggcgcgctg gctcacgcct17401 gtaatcccag cactttggga ggccgaggtg ggtggatcat gaggtcagga gttcgagacc17461 atcctggcta acacggtgaa accccgtctc tactaaaaat acaaaaaatt agctgggcgt17521 ggtggcgggc acctgtactc ccagctactc gggaggctga ggcaggagaa tggcatggag17581 gcaggagaat ggcatgaacc caggaggcag agcttgcagt gagccgagat cacaccattg17641 cactccagcc tgggtgacaa agtgagactc cgtctcaaaa aaaaaaaaat taaaaataaa17701 taaaaataaa aatagactag tttctccata atcgttttcc ttgctttcat ataaatttta17761 aaaatttatt tcagcctgta tgttagcaat ctctggtcac attacctatt ctcaagagtc17821 actctgttgg gaacatctca ggccttctct aaagtattgc tagcacacca ctctgagacg17881 tgggagtctg tcttcccctg tcgcctccct ccccttacat cttctgatca actccacctg17941 cccattcttt catccttcct agtgactttt tctggttcat cagaacctta cccagtctca18001 aagaaaggag tgtgataagg caaacaactc taaataaaat acagttctgt atctttctat18061 gcaaataagc ctttgttaat ttttatttag tgttgtacca cttatttgaa tgtctgacag

18241 aacctttttt taatgattat ttttggtata taatatagtt tcaatgattt ggatggatta18301 gggaaagcca atctaaaact aagaatatta tgaaggctgg gtgcggtggc tcacacctgt18361 aatcccaaca ctttgggagg tgaggcaggt ggatggcttg aggccaggag ttcaggacca18421 gcctgagcaa catggcaaaa ccccgtctct actaaaaata caaaaattag ctgggcatgg18481 tggtgtgcac ctgtagtccc agctactcag gaggctaagg cacaagaatc gcttgaactt18541 gggaggcaga ggatgtagtg agccgagatt gcgctattac actccagcct gggcgacaga18601 catttagttt tatgttcata cacatacata tactcaggat gcacattaaa ctatcaagag18661 tatttacttt gggagtgaaa atgagggtgt gcaaaggaga tatttcaact tttactcttt18721 ttgtcttttt tttttccttt ttgtggagaa cggggtctcg ctatattgcc caggtaggtc18781 tcgaactcct gggcttaagc tatccttcca cctctgcctc cctaacagct gggattacag18841 gtgtaagcca ccacgcccag cccatctttt attcttttaa atattttcta ttgtttgact18901 ttttaataat attttcatgt tttcccttta taataataga aagtgtatat aatgaactct18961 gctaggtttg tcaatattta cacagaatct cttggtgacc tgctctaatg aaacaataat19021 agtttgtaaa tatattaact cattcaacaa gtattaattg agtgcccatt gtatgccagg19081 gactgtacca ggccctaggg atacaataat gagaaaagat atatatgcaa gagaaaaaaa19141 taattacaca aacataaaat tttagttcta gcccctgtaa tggagaaaga gatacaagat19201 gctctaagaa cctgtattgg gagatttgat ttcatcaggg agttcaggga agtgcttctt19261 ggtaaaatga ggtctgaata tgaatatgag ttaactaagt aaagaaagga gggaacaacc19321 atgcaatttg tgatttattc tcctcagtga gtggaaacag ggcaagggga actggagatg19381 aggctagaga agtaggtagg agtcagatct taaatgccag ctacaagctt tgctttgtcc19441 caatactgtt ggaaaccatt gaaagctttt ttttttcatt ttttatcatt tgtattttga19501 tgttgttttt ggggtggtac atgtcacatc acatttgcat tttaaaaaga ttgctctggt19561 tgtagtgtgg agaatggagt aggagtaggg gtggagtccc agttaactgt agattgactg19621 ttaaaagaat attgcaatat ctgagacatg gttagatggg accagggtgg actagagagc19681 agtgaatgta ttggaaagat atttaggaga tagagtcagc agggcttggt gatggattgg19741 atatggggaa agtatgtaaa agatgacttc tggtgttctg gcctccatag ctgagttgat19801 gatgttgcag gacccgtgtt gcaggcagag cagagaggat gaaaatcatg agttcagtct19861 ttgaaatgct ccagaagaga tgtcaaggtg gccaggcacg gtggctcacg cctgtaatcc19921 cagcactttg ggaggccgag gtgggtggat cacctgaggt caggagttga gaccagcctg19981 gccaacatgc tgaaacgctg tctctactaa aaatacaaaa attagctgga catggtggca20041 gatgcctgta atcccagcta ctcgggaggc tgaggcagag aattgcttta acccgggagg20101 cggaggttgc agtgcgccaa gatcgcacca ctgcactcca gcctgggtga cagagcgaaa20161 ctctgtcaaa aaaataaaat aaaaagatgt caagaatgtg gttggatatt acaagtctgg20221 cctagacaag tgttctgaca tggagataca aatttgagcc atcagatgca gaaaagtgat20281 cactgaaacc ctggatataa atgttatcac ctagaaaaaa gaagagtgag gagaggtctt20341 gaatctagaa taattctagc acttaagagc tcggtagaag aggatgaacc agcaaaagag20401 actgtgaaag aacagctaga gaaataggaa gaaaacagaa gaatgttatg ttggaaaacc20461 aggatgtcat gccaaacgct gccaagacat taagtaagat gaggatcatc tgattttgtg20521 acagtagaga ttagggattt tagcaagtga gagctgtttc agagaataga caggacgaga20581 tgctgatggt gctaatggag actgcaagga gatactcaga aatgtgccca tgacacggaa20641 gagggactac agagggtgag ggtgacagag gacctttttt ttttaatggg agaaacttga20701 gcacatttgc aagggatgcc tgatgggata aagtccctga aaaggcctga gtggctgagc20761 cctgagcagc agtggagggc tggctccagc ttgatagtac ctgcaggcaa gtgctaaagt20821 tttagagcat ttattctcta tgtatttcaa aatattccct ttaccttgtt tagtaaatgt20881 catagcagat caaaggcaaa ttaacctaaa gttctgtcag aattatcatg aatatagaat20941 ttgtgaagat caaattccta tagttttact atttctagaa aagtaatagt tttttgttgt21001 tgttgttttt gagacgaagt ttcactcttg ttgcccaggc tggagtgcaa tggtgcaatc21061 tcagctcact gcaacctccg cctcctgggt tcaagcggtt caccagcctc agccttccaa21121 gtagctggaa ttacaggcac ccgccaccca cgcccagcta attttgtatt tttagtagag21181 acggggtttc tccatgttgg ccaggctggt ctcgaactcc tgatctcagg tgatccgccc21241 gccttggcct cccaaagtgc tgggattaca ggcatgagcc aacccgcctg gccaattttt21301 tttttaagtt ttcattatct ggaattcttt gttgtttttg agatcagaat ctctactaat21361 gatactcaag atgggtgtaa ctacaaaatc tgcagatgtt gaaagaagca aaatggatct21421 gttgaaattt ctcttttttt ctttactttt tttttttttt tttttttttt gagatggagt21481 cttgctctgt cgcccaggtg ggattgcagt ggtgcaatct cggctcactg cagtctcccc21541 ttcccgagtt caaggaattc ttctgcctca gcctcccaag tagctgggac tacaggcacc21601 tgccaccacg cccggctaat tcttgtattt ttagtagaga cagggtttca ccatgttggt21661 cagctgatct tgaattcctg acctcaagtg atctgccctc ctcagcctcc caaagtgttg21721 ggattacagg catgagccac aggcccagcc agaatctgtg ttttaataag atccccaggg21781 ggtatgtgat atgaacgttt gagaagccca agtctaaggc aggataaaca tggcccgagg21841 ggaatctggg catcacctct ttctgtacag ccagggagat gggaatggtt tttacatttt21901 ttaatggttg ggaaaaaaaa agaagactat ttcttgacac attaaaatta tatgaaatta21961 gtatttcagt gtccataaat aaagttttat tggaacacag ctacgatatt cacatattgt22021 ctatggctgc tttggctcaa aaagaattgt tgagtaggca tgacagaaat cgtgtggccc22081 aaaaagccta aaatatttat gatctggccc ttcagaaaac gttctccggc ttgtgatcta22141 aagtgcactg gaagtacatt ctttgtgggt ttccaagtag cttgtacacc aaggttagct22201 gattataaca atgtgttgtt taacgatcct ttatcatatt aaagttgaat taataaggca22261 aaatcagtat ttaaacataa gaattatata ttcaagcttc aaacttgagg aaatgccact22321 ttgacttttt tttttttttt taagatggag tctcactctg tcaccaggct ggagtgcagt22381 ggtgcaatct cggctcactg caacctacac ctcctgggtt caagcgattc tcctgcctca22441 gcctcccaag tagctggaac tacaggcgcg tgccaccaca cccagctaat ttttgtattt22501 ttagtagaga cagggtttca ccatgttggc caggatgatc tcgatttctt gacctcatga22561 tccgcccgcc tcagcctccc agtgtgctgg gattacaggt gtgagacact gcacccagcc22621 ctgactatgt tttctaaatg agatgctggt gaactaaaga gtttcttagt ttaccttttg22681 tttcctaaaa aatgaaatta tggggaaaat aataaaactt ataggataaa aattagtaat22741 actggtaatc tctggagaga gaaactagaa ggggtggggg caagagtata aaggagactt22801 tattaaattc ccttttgtgc ctttcagaat tggatctggg attcaaacat atgggataag22861 tttgctatca ttttccccat aatttcatgt tttaggaaat aaaagataca attgttactc22921 ctcagaatgt tttgtcttat gctgttgtta gatagtttga attaagtctt ctctgtcctc22981 aacacacatg atggttgtcg tcttgctctg agaatcaaag ttcacaatcc ttgctcatct

23161 atcagtacac ctcaaagagc ttttctaact gcaactgttg tgagaaagca cactttgtac23221 tgtatgttca aaaattaaac taaagtcctg cagtttggcc tgtgttataa ttgaaagaat23281 gtggtagttc actaattgaa agatgtgaac ttatttatgt ggtcagaggc atatttgcca23341 gatgttatta acaacccaga agtacgcaag gataggctgc ttactctgac atccagcaca23401 aagctctgcc cctccatatg cccggtgatc ttggcatgcc ttcccacact ccatcaggca23461 gagaccagtg gcagctcaga aggcccagta tgctgttttg attttaaaat ttagtttgct23521 tttgaatatg cagtataatc aaatagatcc aattctgaaa ggcacaaaag ggaatataat23581 aaagtctcct ttctatcctt gtccccaccc ctcctagttt ctctccccag agacaaccaa23641 tatctctggt ttttaaatct atctttccaa agcaatatat gctttttaaa tgtttttttt23701 tgctaccctt ttcctcatca tgaggcagtg tgttcaccaa gtggtaaaat accaggaaag23761 tctttactcc tgtggccaat gagtaatgtc aaagagttag gcaaggacct aaggagtgct23821 tcatggacta gaatggcaat gatattacta ctgttttcac ttttccttat aaaatgacgg23881 cctaagagtt gggaaagata tagtaaaaac agtgttagaa aaacttacct ctacaggccc23941 tgcaccattt gagaatagta ctaattctct ccagagactg ggagaccctg ctcaaggaaa24001 gggaagcctt ctggagccat aagactgtgt ggtcaaccca catatgttcc tggtaatcca24061 cattattcac ctctgtcatc aaaacaacat ttattaacta caggaacatt ggcttgttca24121 tctgaatgta agatgcaagt cttatatttc ttattttgaa aaatactacc aaaattgtta24181 ataggctgaa tatttattag tttgacttta aaaatggaaa tccaaaatct aaaactgaac24241 attaaacatt ctgctttgca ttagaatgat tatctcaagg ccgtgcttta tttttatctg

24481 agaatgtgat gttgatggta tcaattctca caataattct gtgaggaata agcataacta24541 ttgttccatt tctcagatga gaccaagaga ggttacaaga cttgtctaag gtcatagcag24601 ttattaagta gtggagcagg cactcaaagg caggtcgtct gactccacag gctgtttgtt24661 tcctttttta aaatatattt ttttctgcat ccaatcatcc attaaaatat ttattttgtg24721 agacatggtc tcactctgtt gtccaggctg gagtgcagtg gcacaatctc tgctcactgc24781 aacctccacc ttccaggctc aagcagtcct cccacttcag cctcccaagt acctggaact24841 acaggtgagc accaccatgc tggactaatt tttgtaattt tttggtagag atgggttttc24901 gccatgttac ccaggctggt ctgaactcct aggctcaagc gatcctccca ctttggcctc24961 ccaaagtgct gggattacag gcgtgagcca ctggacccag cctattcttt ccactgtatc25021 ccagctgaag agtataggat agagtttccc tgataatttt ttccagcagt tggcttagaa25081 cactcttctc atttcttgga acagtctgtg tttatgccat gctttgtgaa agcaaagttt25141 ctgcagtaat tggtcccatt ctgctttgct ttggtgcatt ctagactctt agttttcatt25201 atcgtaatca taatgggaag aaagtaaaac attgggatgt aaggaagggg cgaatcatat25261 agtgagtcac aagataggat atttttcctc ttctaggcag cacaaattat tgaaaatgaa25321 aaagaaattt ctcccaatat gtttcaggtt tgggaatatg atactgccag taaatctttt25381 tttccctatg acataattta tattagaggc agatctattt acttacatcc cgttagtcag25441 gacttctcaa ttccttgggc tgttaattac ctccctgctt taatgctgat tcaaatcact25501 ctcagacctt ttcttcatgc cagactccct taagtgccag cagccagaag ttagcagttc25561 ctcaatgaag aaaacacact tagaaacaag acttagacac agattgattc ccaatggaaa25621 agagaaaaac aataaaggtt actgtaattc aaaaaggaaa tttgataata aaaaatccca25681 aacacccaca cataagtgtg agaaaaacaa tacggttgtt aagccctgtg gcagcttttg25741 aaaaactatt aagtagtatt tggaaccagt agtgaagtgc aaatgaagta gcactctttt

26041 atattttgtc tgtagattct ctgaattcat cttttcttca acaagtattc atggtattta26101 cctgcggtgt gccaggagct agaaattcag agatgagtta gctcaaaata aggaagaagg26161 gcatttaaag aattatagtc agtgaacttt ggctagtaga ggtatgtgtt ttgctttctt26221 cccagtgata aaggtaatga aaactaagag tctagaatgc accaagatag tttttcagaa26281 gtgttatgga aacacagaat gacttgtgtg cctggggagg tgaagtcaat gaaaatgtca26341 cagaaaagct gttttcttaa gaaaagtttt tacttttgag agaccaggtc tcactctgtt26401 gcccaagctg gagtgcagtg gtgcaatcat agctcactgt agccttgatc tcctgggcac26461 aagcaatctt cctgcctcag cctcccaagt aactcggact acagacacgc accaccatgc26521 ctggctaatt ttttcctttt ttttttttgt agagatgggg gcctcactat gttgcccagg26581 ctggtcctga acttctggcc tcaagcaatc cccttgcatt ggcttcccaa acccccggca26641 ctacaggcat gagccactgc acacagccag aaaagtctta cagaatagtg ggaaactagg26701 ctgggcaagg tggctcacac ctgtaatgcc agcactttgg gaggccgtgg tgggtggttc26761 acctgaggtc gggagttcga gaccagcctg acaagcatgg aaaaacccca tctctactaa26821 aaatagaaaa ttagccgggc gtggtggtgc atgcctataa tcccagctac tcaggaggct26881 gaggcaggag aatggcttga acctgggagg cggaggttgc attgagccaa gattgcacca26941 ttgtactcca gcctgggcaa caagagtgaa actctgtctc aaaaaaaaaa agaaaaaaag27001 aatagtggga aagtgctgag tacagaagag gggagaaggg cattctagac agaggaatac27061 atatcagaga cttgagatac atcctatgtt tagggaacta tcaatagata tggctcaaat27121 taagatctac agttgatcct tggataactt gggtctgaac tgcgcaggta cacttaaatg27181 cagattttct ttcacctctg ccattcctga gacagcaaga ccaacctttc ctcttcttcc27241 tcctccttag cctattcaat gtgaaaatga tgagggtgaa gacctttatg atgattcatt27301 tccacttaat gaataataaa tatattttct gtttcttatg attttcttaa cattttcttt27361 ttctttcttt ctttctttct ttcttttttt tttttttttg cgatgcccag gctggagtgc27421 aatggcacaa tctcagctca ctgcaacctc cacctcccgg gttaaagcga ttctcctacc27481 tcagcctccc aagtagctgg gattacaggc gcacaccacc atgccccggc taatttttgt27541 atttttagta gagatggggt ctcaccatgt tggccaagct ggtattgaac tcctgacctc27601 aaatgatcca cctgccttgg cctcccaaag tgctgggatt acaggcatga gccactacgc27661 ctggccaaca ttttcttttc tctagcttac ttaattgtaa gaatacagtg tgtaatatat27721 acaacacaca aaatatgtgt taatcaactg tttatattat tggtaatgct tctgatcaac27781 gttaggctat tggtagttaa gatttgggga aatcaggcca ggtgcagtgg ctcacgcctg27841 taatcccagc attttgggag gccaaggcgg gcggatcatt tgaggtctgg agttcaagac27901 cagcctggcc aacatgatga gaccccgtct ctactaaaaa ttaaaaaatt agccaggcat27961 gatggtatgt gcctgtaatc ccagctactt gggaggctga ggcatgagaa ttgcttgaac28021 ccggaaggtg gaggttgcag tgatccaaga tcactgcact gcactccagc ctgagtgaca28081 cagtaagact ccgtctcaaa aaaaaaaaaa aaaaaattgg ggggaaatca gaagttatac28141 ttggatcttt gactggatgg gagttggcta gatgggttgt caaggtcaaa tcataaaata28201 atttagcata ttctactaag tagtttgcat tttatcctat aggcagccag tgaaaacttt28261 taagcaggag agtaaggtcc attttagatt gcattccaaa tatgattact ttacatatat28321 gttttggtaa aggtttggaa agctactgct gggctgtggc attggaggta ttagagagaa28381 ctattacttt ttattttgat tacttatatg tttctttttt tatagcaaat atcatttatt28441 ttgtctttat tgtctttttc ctctcctttc ctgttttcta ttatagtttt ctgtatttct28501 cttttttttt ttgctctact cgtttagaag ttatatattc catttatatc cttttagaga28561 ttagccttaa cttttagcat gcatacttat caaagtttaa aagtaattca cttctctatc28621 ctcttcctaa ctgtaccaaa atcttacaaa ctgttcatct cccatcaccc tcctcccatc28681 ttccatgttg ctgtctagta ctttaattcc accttgtttt taattccctg gtggtcattt28741 attttatagt caatctttat ttagattaaa aaaagtgttc accagagtct ttgttcacca28801 ttgcctcttg catcctactt attctgtgtt cttttgggtt tttttttttt tttttttttt28861 ttttttttac tgaagtaaat ctcttacaag atcttttatc cacaaacaga tcttgagaca28921 aaaaatacaa aaatataaaa ataaatagaa gttattttag taagagtccg tgggccaggc28981 atggtggctc atgcctgtaa tcccagcact ttgggaggcc aaggcgggca gatcacctga29041 ggtcaggagt tttgagacca gcctggccaa cgtggtgaaa ccctgtctct accaaaaata29101 caaaaattag ctgggcatgg tggcaggtac ttgtaattcc agctactcgg gaggctgagg29161 caggagaatc acttgaaccc gggaggcgga agttgcaatg agctgagatc acgccattgc29221 accccagcct gggtgacaag agtgaaactc tgtctcaaaa aaaaaaaagt ccgtgaatga29281 taagctgtct tttaaagtat ctttattttg tcttcatact tgcatgatta tctagctaaa29341 aacagaattt cacattgcca gttatccccc ccgcattttg aaaatatttc actacttttg29401 actttctatt ttattttatt ttatttcatt ttttgagaca gagtctcact ctgttaccca29461 ggctggagtg caatgggatg atctcagctc actgcaacct ccgcctccca ggttcaagcg29521 attctcctgc ctaagcctac aaagtagctg ggattacagg tgcccaccac cacacccagc29581 taatttttgt atctttagca gagatggggt ttcaccatgt tggccaggct ggtctcaaac29641 tcctgacctc aggtgatctg cctgccgtgg cctcccaaag tgctgggatt acaggtgtga29701 gccactgcac ccagcctgac tttctgtttt agccattgaa gtccttagtc aatccaatta29761 ttactctgta ggtaatctga ttttccctct ggttgctttt acgatggttc actttgtctt29821 tggtgttctg caatttccct atgatgtatc tttgtatgga tttattttca tttccttctg29881 gagattcact atgcttcttc agtttggaaa gtatgcagtc attatcacct caaacgtttc29941 tttcccattc tctctcttgt ctcttggaaa ttttgtcaga tatctactgg gccttatctt30001 tctactctgc atgcctcttc aactctcttc catatttccc attgctttct ctctgtgata30061 cattctgtga aatctcctca cttatcttcc agtgaactaa agtatctctt catcttctgt30121 ttaattatgt gtttagctta tccactgagt tttttatttc agtgactaaa ttttttattt30181 caatggctat attttttatt tccagaagtt gtgttttttt ccccaaatct cactcttcct30241 tttttatggt gtcctgcttt tttcatgatt gttccttctc ctttatcagt agctttcttg30301 atttggagca caggcaaatc cacacccacc tcatctagaa gtctgaagtg ctttagtaat30361 atctaaaata gtaaaatcta aaattagagg atagatgtag tagataaatt gggggatgga30421 tttatgtggg aagatttgag tcagggagag cagctgacgg gttattgcag tggttaaggt30481 aagagaagat gaggattgga ctgagtcagt agcagaggga gtagagagga gtgacatatc30541 tgagacagaa tttgggaggc agaagcaaga ggacttggtg agcaatcaaa cgtgaaagga30601 ataaaggaga aaacaaaagc aacagtgatt ctgagggttg tagcatggat ggctggaagg30661 atggtggtgc cattgacaga gaagggacac agatgaggag gaacaaacct gcctaggagt30721 tccatcgtga aggtgggttt agacacagtg aatgtgcctg caggtggaat gcccagtgga30781 taggcacact taaaagatct ggtacttgga aaataaatct gggctgtaca tatctatctg30841 agagttaaca gtatatatat attgatagta tcagaagctg gagaagggcc ttgaggaagt30901 gaggttgtcc agggagaata tggagagtga aaaggaagag acaaaagagt ggagcctgtg30961 gaacatcagc atccagggtg cgtatgcttt ggaaaaggtc tggaaagctc tgctgaaaag31021 aggaaattct aaggatggct gaaaatactt ggttagagag ggaggaggaa aaccaggaaa31081 aagcaatatt gccaaaacta aggaaagttt taagcaagga gggaatggtc aataacggct31141 atgaagtaga gcctgaaacg ctggacttga ctgtcatgag gacattagtt gtcttgtgga31201 atcatttcct ggggatgagg tggggcagaa gataagagtg cagtgagtca ggagtgcgtg31261 aagcaggagg aagtgaagct aagtagacaa ctctttcagg aactttggct acacacagaa31321 gaagagagat ggggtgagag ctaaaggtta gaagtggttg ggaagtgtgg ggtgggattc31381 aaggctgagg aaaggtgttt gtttgcttta agacaaaaaa gacgtaagca ggtttgcagg31441 ccaaggggga aacattcaag aggaagagca gctgtcagtc agctcttggg tcatcttggc31501 tttgcccttt ccagaatcgc ctttggatca gacagatcca aagatttgct ctgcttccag31561 caaagctctt tcctccccac agacatgctt tctctaagac ccctgctcca aattgtccta31621 aatccaaaag ataggatctg ttagagtctt ttattgtaat tcaatcccta atatgtaacc31681 agaaaattat aagaaaaact tttcctgaag gaacttaaaa gactactgtt aaggagtttg31741 cttcaaatgg aaacaaacct ctggtttcat aaaatcttga ttatcctatg tcaggctgat31801 caggagagac tttgtgtgat tctggaagaa gctggaacag ttttggtgtt ttgaggaggt31861 agctatttga atcctagcag ttgaaagatc attaatacat ccttctataa agctctatct31921 tctagcctgg tgaggaggtg gaggctgcag tgagctgaga tcgcgccatg gcactccagc31981 ctgggcaaca acgagactac gtctcaaaaa attaaaaata ataaataaat acggccaggt32041 gcagtggctc acaactgtaa tcccagcact ttgggaggcc gaggcaggcg gatcatctga32101 ggtcgggagt tcgagaccag cctgaccaac atggagaaac cctgtctcta ctaaagatac32161 aaaattagcc aggcgtggtg gtgcatgatt gtaatcccag ctactcagga ggctgaggca32221 ggagaatcgc ttgaacccag aaggcagagg ttgcggtgag ccaagatcgt gccattgcac32281 tccagcctgg gcaacaagag tgaaactcca tctcaaaata agtaactaac taactaaata32341 aataaataaa aaataaaata aaaatattaa attatccagc attaaacaga ccattggaat32401 ataaaatgga ctgagatgag acccatcacc ccttcactaa gcttacaaac agatgtttta32461 ccatcccaga tgtacaaaca caagaatgtt tgctctccat ccacactaag ctttgtcagt32521 ggattgagtc ttatgagacc atctttctgt ttaggtttgt ttgggaaaac atgtgatttt32581 taaattatat gtaatatagt atctactacc tagctcttga taactgtgcc catttatagg32641 catggcagac tagagtatag atgaaggagg gaagagaatt tcctgagtct ggttttaaaa32701 gaaaaggtac catgttgtaa tcttaggagc agatttacac atagcctttt ccaggagtaa32761 agaaatgatt ttcttataaa gaaatcatta aatgtgcaac aggttttttt ctttttacct32821 ccagaggtta aacaaattgg tagcatgaaa tagtgtctgc tttaaatttc tttaagcttc32881 ttttgtaaat attaatatcc acagggatat aactattgaa aaggttaccc cagggacaga32941 gacatgcaag ggaggagaaa tttctcaaca aagggcctca gaaaataaac tttctgacct33001 gaagggaatt gtccccaaaa agcccgtggg aactaaagtg actgtttact tatgtttatg33061 atatttaagc aagtgttata ctgagcagtg gttgtgttga tggttgttgt gtatttattc33121 tcagtttgtg tgtaatatta cgcttgttct ggctaagatt acacacacaa acagacagcc33181 ttttagtgag aagccttcct gatgttttga attggttgaa tcatcaaggc tactctcacc33241 agctacataa cctcattagt tgagaagcta tctctggagg aagtctcagt agctaaatat33301 attctgtgtt caataagagt tctattcagt tgcttggttt tttcatattc aaagtgcctt33361 acaacaaata aatttgtacc caagtgctgt attgctggaa ctctttcttt caggaggcat33421 gatttaaaga agaaaattga gatattttgt aaactatcaa agggcaagta aaacataacc

33541 aagcttttcc tttattttgg ggtttcacac agaagcacca cacataaaag gcttcgttgt

33721 taagcccttc aacatagcag tgatcaaaca cctctgcatg cagagccctg gcagggctgc33781 cgggattaca aaagaggcag agggtacatt tactacttga ctcttggaga atacaattta33841 gatcaagaaa ctgaatatac ccttttaaaa atcactttta caaaaacaaa atcccaacac33901 aaaaatttga ctatagtgct gcctgaccta tatgagtgga ggaataactc ctagaggaga33961 tgcattttga gatgaatttt gaatgagctg gggaacatgg gttcatagca tggaaagggg34021 aattcactat agggagagta aacagcctgt gcagagaggc agaacagaaa caagttagtt34081 atatgagaag cagcaaagag tatcaagaaa ctaggcaggt aagagaacag aaacctagaa34141 ggtcacagca aaacctttgg atttgataca gaaggcatta gaattagcaa gcgttctttt34201 tttatctttc caactttcat tttaggttca gggggtacat gtgcactttg taacatgggt34261 gagttgcatg tcgcaggggt ttggtgtaca gattattttg tcacccaggt aatgagcata34321 gtacccaata ggtagttctg atcctcaccc acctcccacc ctccaccctc aagtaggccc34381 aggtgtctgt taccccttct ttgtttctat gtgtattcaa tgtttggctc ccacttttaa34441 gtaagaacat gcagtatttg gttttctaat tcacttagga tgatggcctc tagctctatg34501 tttttgcaaa ggatgtgatc tcgttctttt ttatggcgac atagtattcc atgatgtata34561 tgtaccacat tttctttatc caatccacca ttgatgagca ttgaggttga ttccatgtct34621 ttgctattgt aaatagtgct gcagtgagca taacacatgc atgtgtcttt atggtagaat34681 gatttctgtt tctttgggca tattcccagt aataggatta ctgggttgaa tggtagtttt34741 gctttaagtt ctttaagaaa tctctaaact gttttccaca gtggctgaac taatttatat34801 ttcccaccag cagtgtataa gtgttctgtt ttctttgcag cctcaccagc atctgttatt34861 ttttaacttt ttaataccag ccattctgac tggtgtgaga tggtatgtca ctgtgatttt34921 gatttgcatt tctctaatga ttagtgacat tgaacatttt ttatatgctt gttggccatg34981 tgtatgtctt cttttgagaa gtgtgtgttc atgtcctttg cccatttttt aatagggttt35041 tttggttttt gcttgttaat ttatttaagt tccttataga ttctggatat tagttcctta35101 tatagattct ggatattaga cctctgttgg atgcagtttg caaatatttt ctcccattct35161 gtaagttgtc cgtctattct gttgatagtt tcttttgctg tggagaagct ctttagttta35221 attaagtctc acttgtcaat tttcgtttta ttgcagttgc ttttagagtc tttaccagga35281 aatctttgcc aaggcctacg tccagaatgg tatttctgag gttttcttct agggtttttg35341 taaaaagatg gaatgcgccc aggcgcggtt gcttacgcct gtaatcccag cactttggga35401 gaccaaggcg gatggatcac ctgaggtcag gagttcgaga ccagcctggc caacatggtg35461 aaaccccatc tctactaaaa acataaaaat tagccaggcg tggtggcggg cacctgtaat35521 cccagctact caggaggctg aggcaggaga atcacttgaa accaggaggt agaggttgca35581 gtgagccaat atcacgccac tgcactccag cctgggtgac agagtgagac tccatctcaa35641 aataataata ataataataa taataatgaa gatggaatgc ttcatgaatt tgcatgtcat35701 ccttgtgcac ggaccatgct aatcttctcc gtatcactcc aattttatta tatgtgctgc35761 caaagcgaac accattgagg gttcttgagc agggtaatat gatacaatat agaaaatggg35821 gagctgggtg aggccctgtg aggaaggtgc ctgcttctcc ttcaccttcc gccataattg35881 taagttttct gaggcctccc cagccatgca gaactgtgag tcatttaaat ggtctttcct35941 ttataaatga ccgagtcttg gtcatttctt catggcagtg tgagaacaga ctcatactcc36001 tctatcacca aatgtcatca ttgccacctt caaaatgtat cttcggtccg tctgattcag36061 tctgtctggc tcctccctca tttttgccac tgttgagctc tcttacttag ttcttcacat36121 ccactctggc tccctttcaa gctgctcttc tctcagcacc cagaggaatc tttttgaaac36181 atccctccaa gcatgttgtt cttctaacta aaatgtgtca atgatgaccc attgctcttg36241 ggatagaaat gtgatttttt actgtggccc acaagggtct tcgtgatttg gcccctgcct36301 gcctctccag tgtcacgtca tatcactctc caccttgttc tttacatccc agcctgcttt36361 tttggtcttc tgcttcctgt gtgctaaaca tttgtcagcg tcggggcctc tgcactggcc36421 ttctcctcta cctgaaacac ttttctctcc ttcttcaatt agccacgtct ttcttaacct36481 aaagtctcag cttgaatact actttgccag gaaagccttc cttaagctcc taaactgggt36541 ttggtcttcc tattatatat tcatatcctg tattttatag cacctatatt tatcatttaa36601 attattgtat aattatctgc ttcatgtttg tctcccctat tagaccataa gcgccatgag36661 gatggaaatt atctgtctca ctaactgcct gacaaagctc ccaatacaaa gtatatactt36721 aataaatatt tgttgactgg gtaaattgtg cataaaggca ggttataaca atatgagagt36781 ttagattagg aagattatta tggaaattag aaaaaaagaa gcaaataaaa tgaacattgt36841 aaggaaacaa tcagagattg ctattggacg atccatggag aaagataacc ccaacttcac36901 aaacccataa agagtaggca aaaatactgc tcttgcctct tggagaaaaa ctctgaaagc36961 cagatcctaa caagttaagg aatgcctcct acagagatta gaaatacaaa tactccgtaa37021 gaaaaagctg agagacagaa ggtcagtcct gaagccttag tttcatccat ggtcataagc37081 actaatttat ttccctcttt tttttttttt tttttttttt ttgagaacgg agtctcgctt37141 tgttgcccag gctggagtgc agtggcgtga tctcggctca ctgcaacccc cgcctcctgg37201 gttcaagtga ttcttctgcc tcagcctccc aagtagctgg gactataggc gcgtgccacc37261 acacccggct aatttttgta tttttagtag aggcgggatt tcaccatatt ggccaggctg37321 gtcttgaact cctgacctca tgatctgccc gcctcagcct cccaaagtgc tgggattaca37381 ggtgtgagac accatgcccg gcctataagc accaatttca agcaggatta aagcttcatt37441 aaagaagcct aagaaacatg ttactttttc cagatatttg gcagtgaccc aaggtcagca

37741 gtttattttc tctttctttt ctggctttta ctatgcaact gtaatctgtc ttaacctttc37801 caaaatagga gagttttgca aaatctgcac tggaagtttc acataaagcc tcacccattt37861 cattttccag ctcaaatcca aaatggaact tagactgaac tgtcaggtca cctacataat37921 ggctttgtat tttaaaactt aacacatgta cagacttatg taccctgaat atctattgcc37981 tgctggctct aggtttcata atatgcagtt ctttctgaag ctactgaaag ttggtagaag38041 cacagcaaat tcactgttgg caatatagag cctcaacttc cagtttcttg atagtacctg38101 ctggaaacgg tgatctgtga ctgtagctgg ccatccccaa ggatcttgag ttatggtttg38161 aggatcatca gacagggacc caagcttggg gatctctaca ttatgctttg gccctaatta38221 cccagcaacc tgcaaactac aatgtggata atgcagaaaa ggcacagaat ggcttaaatg38281 aagaacttct gtccttgtta taatcttcag tgaaggagaa catggagaac ataacaggaa38341 tgtccctgtt gctgctttcc aaaatctttc cttttttttt gttttttgag acggagtttt38401 gctgttgttg cgcaggctgg agtgcaatgg tgcgatcttg gctcaccgca acctctgcct38461 cccgggttca agcaattctc ctgcctcagc ctcccgagta gctgggatta caggcatgcg38521 ccaccacgcc cggctaattc tgtattttta gtagagacag cgtttctcca tgttggtcag38581 gctggtctcg aactcccgac ctcaggtgaa ctgtccacct tggcctccca aagtgctggg38641 attataggca tgagccactg cgccctgccc aaaatctttt cttttgctaa cctttccctg38701 ctaagggata taaaatcttt tcttgcatgg agttttcagg tactaatgtt gtctgactca38761 actagctgat tttacttcat taaaacctct ttgtttcaca aagaaaaata tgaagaaagt38821 ttttctccat tgctgagagc tgggaatcta gacttaaagt tctgagtccc cttggatcct38881 taaaacaatc tgtaatctct attgaatcca gaagttagaa atctaaacag attataacag38941 ccagactttg gaaatatttg tatgctacct acattttcaa tttttttttt ttgagacgga39001 gtctcactct gtcgcccagg ctggagtgca gtggcatgat cttggctcac cacaacctcc39061 gcctcccagg ttcaatcaat tctcctgcct cagcctcctg agtagctggg attataggca39121 tgcgccacca cacccggcta attttttttt gtatttttag tagagacagg gtttcaccat39181 attggccagg ctggtctcaa actcctgacc ttgtgatccg cctgccttgg cctcccagag39241 tgctgggatt acaggtgtga gtcacctcgc ccagcctgta tgctacctac cttttcatat

42781 tcattcagca aatcttcagt gagttccttt ctaaatgtat tgtatcagaa cttgtggggg42841 atataaagaa aaaaaagagg ttatcacatg gcttaggatg tttccaaccc ttggatcatt42901 ttcaccccag gcccattagt tacagagcac tcagtcttac ccagaagcat cggatatagt42961 ccaggcaacc ggatccaagc gtgtgggaat cgcagtgtgc tgcatgtggg aatctatcag43021 gccaggatga ttcaagcagc agcctgcaga agtgggattt ggccagcctt gcctgactct43081 ggccttccaa cttgctctta tctacttcca aagtaaggaa tgccccatga tgggccgagc43141 agtccaaaaa aagtggcttg gagttagcta ccgcaattag cagtttctca ttattcaaca43201 ctgcattacc tctttttcat attaactgca gaattttatt tttatgtatt tattatcctt43261 ccaaacccag tgttgtagga agaatattac atataaatga taacttagga atcttgtcaa43321 gtttttgttt cttcagcact aagtagctta ttttctaggc aggtcttaat tctaaatgta43381 acatgcttga aaaaaacact attgaaagga tccgtctctt cagcatagta tttaatatac43441 atatcaggca atcaccatct caagcatgat tagactcaag tgctgccctc catcgtgtga43501 gggatgctgg gagactggaa gcttagactt tagctcttta agttggtgca aaagcaattg43561 cagtttttac tattaataat aataattgcc tagccattgt gcagtacctt gcaagagctc43621 tttggcaaat gaaagaataa ttcaaagcaa agtaggccag atataaaaca cttccaaatc43681 acatttcaaa attgttcttt catgtcaact aggtgcatgt ctgctaaggg agtttttgca43741 aggacaggtg gatagtgtga ggcctttaca ataatcacat caccatctgg tactatctgt43801 atggcagagc agttctgatg tagttgtagt attatagatc attacagagt atgccagtca43861 ttccatgatg gaactgtgac tggcagcctt cattagccca aagactgatg caacagattg43921 ctttgagaaa tagttttgaa gcacatccat gaaacatatg atcctatggg ttccagcatt43981 gatttctttg aggtatcatt ttacacttca gtttttcctg gtagagtatt tttcagagat44041 gtctcttctg aggctaaatt aggaatttct gttccttatt caagttccaa gaattgtgcc44101 tttcctgccc tctgcccaaa ctggaagata gaaaccgtgg gaaaaaagtg tctttacaaa44161 ctgcactgtt atctaacttg gtttatttag catattgagg aagactttca catccatgga44221 atcctagttc tattaattct ctccaagcta caacagttgt ttttttgttt gtttttttcc44281 gagtttctcg gttccgccag caaggcaggg gtgggaggtg ggcacccctg ataccaaggc44341 tgacaggtag tgagttgatg tggaacttct gtttcctcct gttcagttca ggttctctct44401 ttctgatact tacccctcct caaataggga aacggagaga ggggaaataa gaagatgagc44461 cttaataggg tttacttaat tggggtcata aagattctaa aaagtatgca ttcctgcagt44521 tctgttctag gcactgtaag agctgaccag aaagagagct tctaccctta taaccttcat44581 ccaactcagt catgcctggg aaggatgttg gtgctaattt tagacacaga gactaagatg44641 gacaaaggac aagtatcata aagcattttt ggagccagga acagatttaa gttccttgcc44701 tcactgctgc tcttactcca aaccacacaa ttgtacccaa gtgaattgct gttagaattg44761 gtgcctttg

SEQ ID NO: 2 below corresponds to the reverse complement ofchr14:75500097-75500218 that shows the sequence in the direction oftranscription, where Exon 7 of MLH3 is underlined:

5′-aagttcacaa tccttgctca tctagGTGGG AACCTGCTCGTGCTGGTGGA TCAGCACGCT GCCCATGAGC GTATACGTCTGGAGCAGCTT ATCATTGgta aggatctgtt tgcagccaga aa-3′

The protein MLH3 (as half of MutLgamma) works downstream of MSH3 (halfof MutSbeta) to cleave DNA near bound MutSbeta. MSH3 has variableexpression, and is often absent in tissues that do not exhibit somaticrepeat expansion. When ectopic expression of MSH3 is introduced, therepeat expands (A5). In one embodiment, MSH3 expansion can be inhibitedby inhibiting the next step in the process, DNA nicking by MutLgamma viaMLH3 isoform switching. While MLH3 has not been identified as a majorcontributor to cancer in humans (A6,A7) and MLH3 knockout mice werehealthy and showed no susceptibility to morbid cancer in the first 9months of life (A8), longer term studies have shown that complete lossof MLH3 throughout life increased the propensity for late onset tumors(A9) which was modified by loss of other tumor suppressor genes (A10).

Oligonucleotides

“Oligonucleotide compounds” of the invention can includeoligonucleotides, e.g., Antisense oligonucleotides (ASOs), spliceswitching oligonucleotides (SSOs), siRNA, shRNA, and the like as well asmodified nucleotides discussed herein that are incorporated into thesame. ASOs are single stranded nucleotide molecules that arecomplementary to a target nucleic acid sequence. For example, “targetnucleic acid” and “nucleic acid encoding a subunit of the MMR system”encompass DNA encoding a subunit of the MMR system, RNA (includingpre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived fromsuch RNA, as well as DNA or RNA sequence described herein that furtherencompasses noncoding sequence. In one embodiment, the target sequencecomprises a nucleic acid sequence encoding a subunit of the MMR system(e.g., MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2), where the nucleicacid sequence includes but is not limited to sense and/or antisensenon-coding and/or coding sequences associated with a nucleic acidsequence encoding a subunit of the MMR system.

Hybridization involves hydrogen bonding, which may be Watson-Crick,Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementarynucleoside or nucleotide bases. For example, adenine and thymine arecomplementary nucleotides which pair through the formation of hydrogenbonds. Hybridization can occur under varying circumstances.Complementary, as is understood by the skilled artisan, refers to thecapacity for precise pairing between two nucleotides. For example, anoligonucleotide and the DNA or RNA are complementary to each other whena sufficient number of corresponding positions in each molecule areoccupied by nucleotides which can hydrogen bond with each other. It isunderstood in the art that the sequence of an oligonucleotide does notneed to be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. For example, an oligonucleotide can hybridizeover one or more segments such that intervening or adjacent segments arenot involved in the hybridization event (such as, e.g., a loopstructure, mismatch or hairpin structure). Thus, “specificallyhybridizable” and “complementary” are used to indicate a sufficientdegree of complementarity or precise pairing where stable and specificbinding occurs between the oligonucleotide and its target nucleic acid(e.g., the DNA or RNA target).

In one embodiment, the specific hybridization of an oligonucleotidecompound with its target nucleic acid interferes with the normalfunction of the nucleic acid. In one embodiment, an oligonucleotidecompound is specifically hybridizable when binding of theoligonucleotide to the target nucleic acid interferes with the normalfunction of DNA, the normal function of RNA, or the normal functionand/or expression of the product encoded by the target nucleic acid,causing a modulation of function and/or activity. In one embodiment, anoligonucleotide compound (e.g., an SSO) can cause an intron to beretained. When an intron is retained, for example, the mRNA isde-stabilized and subsequently degraded. Thus, intron retention mediatedby an oligonucleotide compound, such as an SSO, can lower expression ofthe target gene just like shRNA or siRNA.

The DNA functions to be interfered include, for example, replication andtranscription. The RNA functions to be interfered include, for example,translocation of the RNA to the site of protein translation, translationof protein from the RNA, splicing of the RNA to yield one or more mRNAspecies, and/or catalytic activity, which may be engaged in orfacilitated by the RNA. The overall effect of such interference withtarget nucleic acid function is modulation of the expression of anencoded product or oligonucleotides. In one embodiment, the modulationis a decrease or loss of the activity of the encoded product. In oneembodiment, the modulation is a decrease or loss of expression of theencoded product.

The oligonucleotide compounds described herein comprise about 70%, orabout 75%, or about 80%, or about 85%, or about 90%, or about 91%, orabout 92%, or about 93%, or about 94%, or about 95%, or about 96%, orabout 97%, or about 98%, or about 99% sequence complementarity to atarget region within the target nucleic acid sequence to which theoligonucleotide compound is targeted. For example, an oligonucleotide inwhich 18 of 20 nucleotides of the oligonucleotide compound arecomplementary to a target region, and would therefore specificallyhybridize, would represent 90 percent complementarity.

In one embodiment, the oligonucleotides are specific for polynucleotidesof a subunit of the MMR system (e.g., MSH2, MSH3, MSH6, MLH1, MLH3,PMS1, or PMS2), which includes, without limitation, non-coding regions.In one embodiment, the oligonucleotide is an antisense RNA molecule. Inone embodiment, the oligonucleotide is an antisense DNA molecule. In oneembodiment, an oligonucleotide targets a natural antisense sequence(natural antisense to the coding and non-coding regions) of a subunit ofthe MMR system (e.g., MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2). Inone embodiment, the oligonucleotide is an antisense RNA molecule. In oneembodiment, the oligonucleotide is an antisense DNA molecule.

The oligonucleotide compounds discussed herein can also include variantsin which a different base is present at one or more of the nucleotidepositions in the oligonucleotide compound. For example, if the firstnucleotide is an adenine, variants may be produced which containthymidine, guanosine, cytidine or other natural or non-naturalnucleotides at that position. The base substitution can be done at anyof the positions of the oligonucleotide. The oligonucleotide compoundscan then be tested using methods described herein to determine theoligonucleotide compound's ability to inhibit expression and/or functionof a target nucleic acid, such as a subunit of the MMR system (e.g.,MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2).

In one embodiment, homology between an oligonucleotide and its targetnucleic acid sequence (e.g., the nucleic acid sequence of a subunit ofthe MMR system such as MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2) isfrom about 50% to about 60%. In some embodiments, the homology is fromabout 60% to about 70%. In some embodiments, the homology is from about70% to about 80%. In some embodiments, the homology is from about 80% toabout 85%. In some embodiments, the homology is from about 85% to about90%. In some embodiments, the homology is about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,about 99%, or 100%. In one embodiment, sequence identity between anoligonucleotide and its target nucleic acid sequence (e.g., the nucleicacid sequence of a subunit of the MMR system such as MSH2, MSH3, MSH6,MLH1, MLH3, PMS1, or PMS2) is from about 50% to about 60%. In furtherembodiments, the homology is from about 60% to about 70%. In furtherembodiments, the homology is from about 70% to about 80%. In furtherembodiments, the homology is from about 80% to about 85%. In furtherembodiments, the homology is from about 85% to about 90%. In furtherembodiments, the homology is about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or100%. In one embodiment, complementarity between an oligonucleotide andits target nucleic acid sequence (e.g., the nucleic acid sequence of asubunit of the MMR system such as MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, orPMS2) is from about 50% to about 60%. In another embodiment, thehomology is from about 60% to about 70%. In another embodiment, thehomology is from about 70% to about 80%. In another embodiment, thehomology is from about 80% to about 85%. In another embodiment, thehomology is from about 85% to about 90%. In another embodiment, thehomology is about 90%, about 91%, about 92%, about 93%, about 94%, about95%, about 96%, about 97%, about 98%, about 99%, or 100%.

Modifications

According to the invention, oligonucleotide compounds can comprise atleast one region where the oligonucleotide is modified in order toexhibit one or more desired properties. The desired properties of theoligonucleotide include, but are not limited, for example, to increasedresistance to nuclease degradation, increased cellular uptake, and/orincreased binding affinity for the target nucleic acid. Modifiedoligonucleotides can include, for example, synthetic nucleotides havingmodified base moieties and/or modified sugar moieties (see e.g.,described generally by Schcit, Nucleotide Analogs, John Wiley, New York,1980; Freier & Altmann, (1997) Nucl. Acid. Res., 25(22), 4429-4443,Toulme, J. J., (2001) Nature Biotechnology 19:17-18; Manoharan M.,(1999) Biochemica et Biophysica Acta 1489:117-139; Freier S. M., (1997)Nucleic Acid Research, 25:4429-4443, Uhlman, E., (2000) Drug Discovery &Development, 3: 203-213, Herdewin P., (2000) Antisense & Nucleic AcidDrug Dev., 10:297-310); or 2′-O, 3′-C-linked[3.2.0]bicycloarabinonucleosides. Such modified nucleotides includesynthetic nucleotides designed to enhance binding properties, e.g.,duplex or triplex stability, specificity, or the like.

An oligonucleotide compound, whether DNA, RNA, DNA or RNA with modifiednucleotides, DNA or RNA with substituted nucleotides, and the like, canspecifically hybridize when binding of the oligonucleotide compound tothe target nucleic acid (e.g., a DNA or RNA molecule) interferes withthe normal function of the target DNA or RNA. Further modifications caninclude conjugate groups attached to one of the termini of anoligonucleotide compound or to selected nucleotide positions of anoligonucleotide compound, conjugate group(s) added to various positionson the sugar ring, or conjugate group(s) added to one of theinternucleotide linkages. In one embodiment, the interference can causea loss of utility, and there is a sufficient degree of complementarityto avoid non-specific binding of the oligonucleotide compound tonon-target nucleic acid sequences under conditions in which specificbinding is desired. Conditions in which specific binding are desiredinclude, but are not limited to, physiological conditions in in vivoassays or in therapeutic treatment, or conditions in which the in vitroassays are performed.

ASOs comprise a more general grouping of antisense compounds, whichinclude but are not limited to siRNA, ribozymes, external guide sequence(EGS) oligonucleotides, single- or double-stranded RNA interference(RNAi), and other oligonucleotides that hybridize to at least a portionof the target nucleic acid sequences and modulate its function. Theantisense compounds can be single-stranded, double-stranded, circular orhairpin and can comprise structural elements such as mismatches orloops. Antisense compounds are routinely prepared linearly but one ofordinary skill in the art can prepare antisense compounds to be joinedor otherwise prepared to be circular and/or branched.

In one embodiment, oligonucleotide compounds directed to a nucleic acidsequence of a subunit of the MMR system (such as MSH2, MSH3, MSH6, MLH1,MLH3, PMS1, or PMS2) can comprise one or more modified nucleotides. Inone embodiment, oligonucleotide compounds directed to a nucleic acidsequence of a subunit of the MMR system (such as MSH2, MSH3, MSH6, MLH1,MLH3, PMS1, or PMS2) can comprise shorter or longer fragment lengths(e.g., 15-, 16-, 17-, 18-, 19-20-, 21-, 22-, 23-, 24-, 25-, 26-, 27-,28-, 29-, 30-, 31-, 32-, 33-, 34-, 35-, 36-, 37-, 38-, 39-, 40-, 41-,42-, 43-, 44-, 45-, 46-, 47-, 48-, 49-, or 50-mers). In one embodiment,oligonucleotide compounds directed to a nucleic acid sequence of asubunit of the MMR system (such as MSH2, MSH3, MSH6, MLH1, MLH3, PMS1,or PMS2) can comprise modified bonds or internucleotide linkages.Non-limiting examples of modified bonds or internucleotide linkagesinclude phosphorothioate, phosphorodithioate, and the like. In oneembodiment, the oligonucleotide compounds can comprise a phosphorusderivative. In one embodiment, the phosphorus derivative (or modifiedphosphate group) can be attached to the sugar or sugar analog moiety inthe modified oligonucleotides of the present invention. Non-limitingexamples of a phosphorus derivative (or a modified phosphate group)include a monophosphate, diphosphate, triphosphate, alkylphosphate,alkanephosphate, phosphorothioate and the like. The preparation of theexemplary phosphorus derivatives (or modified phosphate groups), andtheir incorporation into nucleotides (e.g., those comprising anoligonucleotide compound of the invention), is well-known in the art.

A number of nucleotide modifications have been shown to make theoligonucleotide into which they are incorporated more resistant tonuclease digestion than the native oligodeoxynucleotide.Oligonucleotides that have been modified to enhance their nucleaseresistance survive intact for a longer time than unmodifiedoligonucleotides. As discussed herein, embodiments of the presentinvention encompass modified oligonucleotides, such as modified ASOsdirected to a nucleic acid sequence of a subunit of the MMR system (suchas MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2). Modifiedoligonucleotides can comprise 2′-O-methyl modified oligoribonucleotides,which render the antisense oligonucleotide resistant to RNase Hdegradation. In one embodiment, modified oligonucleotides comprise aphosphorothioate backbone. For example, the phosphorothioate backboneincreases the stability of an oligonucleotide compound against nucleasesand enhances cellular uptake. In some embodiments, oligonucleotidecompounds can comprise a full length phosphorodiamidate DNA. In someembodiments, oligonucleotide compounds can comprise one or nucleotideshaving a 2′O-methyl modification. In some embodiments, oligonucleotidecompounds comprise one or more modifications discussed herein.Non-limiting examples of modified backbones include phosphorothioates,phosphinates, phosphorodithioates, phosphoramidates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonates (e.g.,phosphonates comprising 3′ alkylene phosphonates), short chain alkyl orcycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages. In one embodiment, oligonucleotidecompounds directed to a nucleic acid sequence of a subunit of the MMRsystem (such as MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2) comprisephosphorothioate backbones.

In one embodiment, the region of a modified oligonucleotide compounddirected to a nucleic acid sequence of a subunit of the MMR system (suchas MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2) comprises at least onenucleotide modified at the 2′ position of the sugar. In someembodiments, the nucleotide having a modification at the 2′ position ofthe sugar comprises a 2′-O-alkyl, 2′-O-alkyl-O-alkyl, or2′-fluoro-modified nucleotide. In some embodiments, RNA modificationsinclude 2′-fluoro, 2′-amino and 2′-O-methyl modifications on the riboseof pyrimidines.

As discussed herein, oligonucleotide compounds can comprise additionalmodifications such as morpholino phosphorodiamidate DNA, locked nucleicacids (LNA), and ethylene bridged nucleic acids. These modifications canrender the oligonucleotide compounds RNase H and nuclease resistant aswell as can increase the affinity for the target RNA. In one embodiment,oligonucleotide compositions of the invention have morpholino backbonestructures (e.g., as disclosed by Summerton and Weller, in U.S. Pat. No.5,034,506, which is hereby incorporated by reference in its entirety).Morpholinos, for example, are commercially available through Gene Tools,LLC, Philomath Oreg.; http://www.gene-tools.com/).

For example, the morpholino backbone of oligonucleotide analogues makesthem resistant to nucleases and proteases so that they are long-lived inthe cell. Some morpholino oligomers can be diluted by cell division andgradually become ineffective after a single dose in rapidly dividingtissues or in growing organisms. In contrast, morpholino spliceswitching oligonucleotides (SSOs) remain active in post-mitotic tissuessuch as brain and spinal cord for several months (A2). In oneembodiment, the region of a modified oligonucleotide compound directedto a nucleic acid sequence of a subunit of the MMR system (such as MSH2,MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2) comprises at least one nucleotidemodified with a morpholino subunit.

A number of nucleotide modifications incorporated into anoligonucleotide (e.g., resulting in an oligonucleotide analog), makesthe oligonucleotide useful for steric blocking applications, such asexon skipping. For example, negatively charged oligonucleotideanalogues, such as oligodeoxynucleotide phosphorothioate (DNA-PS),2′-O-methylphosphorothioate (OMe-PS), 2′-O-methoxyethyl (MOE),2′-deoxy-2′-fluoronucleotides (2′-F), locked nucleic acids (LNA; alsoreferred to as bridged nucleic acids (BNA)), ethylene-bridged nucleicacids (ENA), tricycloDNA analogue (TcDNA), and2′-O-[2-(N-methylcarbamoyl)ethyl]uridine (MCE), as disclosed in Järveret al. (2014) Nuc. Acid Therap., 24(1):37-47 (incorporated by referencein its entirety), can be used to induce exon skipping:

where R can be O or S in the above-listed negatively chargedoligonucleotide analogues. In one embodiment, the oligonucleotidecompounds disclosed herein, such as for example, nucleic acid moleculesset forth in SEQ ID NOS: 3 and 4, comprise one or more substitutions ormodifications. In one embodiment, the oligonucleotide compoundsdisclosed herein, such as for example, nucleic acid molecules set forthin Table 4, comprise one or more substitutions or modifications. In oneembodiment, the oligonucleotide compounds are substituted with at leastone locked nucleic acid (LNA). In one embodiment, the oligonucleotidecompounds are substituted with at least one phosphorothioate (PS). Inone embodiment, the oligonucleotide compounds are substituted with atleast one 2′-O-methylphosphorothioate (OMe-PS). In one embodiment, theoligonucleotide compounds are substituted with at least one2′-O-methoxyethyl (MOE). In one embodiment, the oligonucleotidecompounds are substituted with at least one 2′-deoxy-2′-fluoronucleotide(2′-F). In one embodiment, the oligonucleotide compounds are substitutedwith at least one ethylene-bridged nucleic acid (ENA). In oneembodiment, the oligonucleotide compounds are substituted with at leastone tricycloDNA analogue (TcDNA). In one embodiment, the oligonucleotidecompounds are substituted with at least one2′-O-[2-(N-methylcarbamoyl)ethyl]uridine (MCE). In one embodiment, theoligonucleotide compounds are substituted with at least oneoligodeoxynucleotide phosphorothioate (DNA-PS),2′-O-methylphosphorothioate (OMe-PS), 2′-O-methoxyethyl (MOE),2′-deoxy-2′-fluoronucleotide (2′-F), locked nucleic acid (LNA),ethylene-bridged nucleic acid (ENA), tricycloDNA analogue (TcDNA),2′-O-[2-(N-methylcarbamoyl)ethyl]uridine (MCE), or a combinationthereof.

Charge-neutral peptide nucleic acids (PNA) and phosphorodiamidatemorpholino oligonucleotides (PMO) are further examples ofoligonucleotide analogues, as disclosed in Järver et al. (2014) Nuc.Acid Therap., 24(1):37-47 (incorporated by reference in its entirety),that can be used to induce exon skipping:

In one embodiment, the oligonucleotide compounds disclosed herein, suchas for example, nucleic acid molecules set forth in SEQ ID NOS: 3 and 4,comprise one or more substitutions or modifications. In one embodiment,the oligonucleotide compounds disclosed herein, such as for example,nucleic acid molecules set forth in Table 4, comprise one or moresubstitutions or modifications. In one embodiment, the oligonucleotidecompounds are substituted with at least one peptide nucleic acid (PNA).In one embodiment, the oligonucleotide compounds are substituted with atleast one phosphorothioate (PS). In one embodiment, the oligonucleotidecompounds are substituted with at least one peptide nucleic acid (PNA),phosphorothioate (PS), or a combination thereof.

Due to the uncharged backbone of the morpholino subunit, theseoligonucleotide analogues can bind their complementary target RNA verytightly (A1). Morpholinos work simply by binding their complementarysequence and excluding binding by proteins or nucleic acids. In oneembodiment, binding to a splice donor or acceptor sequence can interferewith recognition of those sequences by the splicing machinery and causeexon skipping. Morpholinos have most often been used for proteinknockdown experiments. A morpholino designed to bind the initiating AUGin an mRNA will block translation initiation by ribosomes. An advantageof morpholinos is the predictable way that they work in differentspecies and different tissues since they are not dependent on accessoryprotein expression such as RISC, dicer, or RNaseH for activity.

In one embodiment, oligonucleotide compounds disclosed herein can bindto a selected target nucleic acid sequence to induce exon skipping. Insome embodiments, masking a donor splice site can induce exon skipping.In some embodiments, masking an acceptor splice site can induce exonskipping. In one embodiment, an oligonucleotide compound (e.g., an SSO)can cause an intron to be retained; thus, when an intron is retained,for example, the mRNA is de-stabilized and subsequently degraded,wherein intron retention mediated by an oligonucleotide compound, suchas an SSO, can lower expression of the target gene. In one embodiment,the oligonucleotide compound is a modified oligonucleotide directed to atarget nucleic acid sequence of a subunit of the MMR system (such asMSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2). In another embodiment, themodified oligonucleotide compound directed to a target nucleic acidsequence of a subunit of the MMR system comprises at least onemorpholino subunit. For example, to induce exon skipping in exons of theMLH3 gene transcript, the antisense molecules are selected from thegroup of SSOs shown in Table 4.

TABLE 4 SSOs for human MLH3 exons 2 through 12. SEQ ID NO: SSO SEQUENCE 7 hMLH3X2ac TCTCTGACTGGAAATAATTGCctat  8 hMLH3X2dnATTCTCAAGTACAACATCCACAGCC  9 hMLH3X3ac TGACACCTGTACTGAGACCctaaat 10hMLH3X3dn tctctgccacccttacCTCTGTTAT 11 hMLH3X4acCATCCACAGTATctagggcaaaagg 12 hMLH3X4dn ccacCTCTGGATAACGGGCAAATAC 13hMLH3X5ac CAGCAACctagaaagactcagcaaa 14 hMLH3X5dngttaatcttttacCTGCATTGAATG 15 hMLH3X6ac TGCTGGAGAACctgtcagacattca 16hMLH3X6dn actccattcttacCTGCCTCGCCAT 17 hMLH3X7acGTTCCCACctagatgagcaaggatt 18 hMLH3X7dn tgcaaacagatccttacCAATGATA 19hMLH3X8ac GAATctattggcagaaagatgaatg 20 hMLH3X8dnacattctcatggtggtactgacCAT 21 hMLH3X9ac GTAACACctaaagagataacctcaa 22hMLH3X9dn taacatctgcagctgtgtcttacCT 23 hMLH3X10acCctgcaaagcaaaaggaaaatcggc 24 hMLH3X10dn tacCTCCAGTTGTTCTCGGATAAAT 25hMLH3X11ac CGGTGGTCTGGAGTAGctaatgcat 26 hMLH3X11dnctatgttgaagggcttacCATGGCA 27 hMLH3X12ac AATGGCCCctaaatgaaagacagaa 28hMLH3X12dn tgctcctgttagtcattaatgtacC

In one embodiment, an oligonucleotide compound directed to a nucleicacid sequence of a subunit of the MMR system comprises SEQ ID NO: 3. Inone embodiment, an oligonucleotide compound directed to a nucleic acidsequence of a subunit of the MMR system comprises SEQ ID NO: 4. In oneembodiment, an oligonucleotide compound directed to a nucleic acidsequence of a subunit of the MMR system comprises a nucleic acidsequence depicted in Table 4. In one embodiment, an oligonucleotidecompound is directed to a nucleic acid sequence (or target complementarynucleic acid sequence) corresponding to a region of interest for any oneof the exons 1-16 described herein for GenBank Accession No.NG_007110.2, or to an intron-exon junction, or to an exon-intronjunction listed with GenBank Accession No. NG_007110.2. In oneembodiment, an oligonucleotide compound is directed to a nucleic acidsequence (or target complementary nucleic acid sequence) correspondingto a region of interest for any one of the exons 1-24 described hereinfor GenBank Accession No. NG_016607.1, or to an intron-exon junction, orto an exon-intron junction listed with GenBank Accession No.NG_016607.1. In one embodiment, an oligonucleotide compound is directedto a nucleic acid sequence (or target complementary nucleic acidsequence) corresponding to a region of interest for any one of the exons1-10 described herein for GenBank Accession No. NG_007111.1 or SEQ IDNO: 33, or to an intron-exon junction, or to an exon-intron junctionlisted with GenBank Accession No. NG_007111.1 or SEQ ID NO: 33. In oneembodiment, an oligonucleotide compound is directed to a nucleic acidsequence (or target complementary nucleic acid sequence) correspondingto a region of interest for any one of the exons 1-19 described hereinfor GenBank Accession No. NG_007109.2, or to an intron-exon junction, orto an exon-intron junction listed with GenBank Accession No.NG_007109.2. In one embodiment, an oligonucleotide compound is directedto a nucleic acid sequence (or target complementary nucleic acidsequence) corresponding to a region of interest for any one of the exons1-13 described herein for GenBank Accession No. NG_008648.1, or to anintron-exon junction, or to an exon-intron junction listed with GenBankAccession No. NG_008648.1. In one embodiment, an oligonucleotidecompound is directed to a nucleic acid sequence (or target complementarynucleic acid sequence) corresponding to a region of interest for any oneof the exons 1-13 described herein for GenBank Accession No. NG_008649.1or SEQ ID NO: 1, or to an intron-exon junction, or to an exon-intronjunction listed with GenBank Accession No. NG_008649.1 or SEQ ID NO: 1.In one embodiment, an oligonucleotide compound is directed to a nucleicacid sequence (or target complementary nucleic acid sequence)corresponding to a region of interest for any one of the exons 1-15described herein for GenBank Accession No. NG_008466.1, or to anintron-exon junction, or to an exon-intron junction listed with GenBankAccession No. NG_008466.1.

In some embodiments, an oligonucleotide compound directed to a nucleicacid sequence of a subunit of the MMR system is a modifiedoligonucleotide. According to the invention, a combination or “cocktail”of two or more oligonucleotide compounds can be provided that bind to aselected target nucleic acid (such as a subunit of the MMR system) inorder to induce exon skipping. For example, to induce exon skipping inexons of a subunit of the MMR system gene transcript (such as MSH2,MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2), the oligonucleotide compounds inthe cocktail are selected from the group of SSOs shown in Table 4. Inone embodiment, the cocktail comprises at least 2 SSOs selected fromTable 4. In one embodiment, the cocktail comprises SSOs comprising SEQID NO: 3 and SEQ ID NO: 4. In one embodiment, the cocktail comprisesSSOs comprising SEQ ID NO: 3 and an SSO selected from the group of SSOsshown in Table 4. In one embodiment, the cocktail comprises SSOscomprising SEQ ID NO: 4 and an SSO selected from the group of SSOs shownin Table 4. In one embodiment, the cocktail comprises SSOs directed tonucleic acid sequences (or target complementary nucleic acid sequences)corresponding to a region of interest for any one of the exons 1-16described herein for GenBank Accession No. NG_007110.2, or to anintron-exon junction, or to an exon-intron junction listed with GenBankAccession No. NG_007110.2. In one embodiment, the cocktail comprisesSSOs directed to nucleic acid sequences (or target complementary nucleicacid sequences) corresponding to a region of interest for any one of theexons 1-24 described herein for GenBank Accession No. NG_016607.1, or toan intron-exon junction, or to an exon-intron junction listed withGenBank Accession No. NG_016607.1. In one embodiment, the cocktailcomprises SSOs directed to nucleic acid sequences (or targetcomplementary nucleic acid sequences) corresponding to a region ofinterest for any one of the exons 1-10 described herein for GenBankAccession No. NG_007111.1 or SEQ ID NO: 33, or to an intron-exonjunction, or to an exon-intron junction listed with GenBank AccessionNo. NG_007111.1 or SEQ ID NO: 33. In one embodiment, the cocktailcomprises SSOs directed to nucleic acid sequences (or targetcomplementary nucleic acid sequences) corresponding to a region ofinterest for any one of the exons 1-19 described herein for GenBankAccession No. NG_007109.2, or to an intron-exon junction, or to anexon-intron junction listed with GenBank Accession No. NG_007109.2. Inone embodiment, the cocktail comprises SSOs directed to nucleic acidsequences (or target complementary nucleic acid sequences) correspondingto a region of interest for any one of the exons 1-13 described hereinfor GenBank Accession No. NG_008648.1, or to an intron-exon junction, orto an exon-intron junction listed with GenBank Accession No.NG_008648.1. In one embodiment, the cocktail comprises SSOs directed tonucleic acid sequences (or target complementary nucleic acid sequences)corresponding to a region of interest for any one of the exons 1-13described herein for GenBank Accession No. NG_008649.1 or SEQ ID NO: 1,or to an intron-exon junction, or to an exon-intron junction listed withGenBank Accession No. NG_008649.1 or SEQ ID NO: 1. In one embodiment,the cocktail comprises SSOs directed to nucleic acid sequences (ortarget complementary nucleic acid sequences) corresponding to a regionof interest for any one of the exons 1-15 described herein for GenBankAccession No. NG_008466.1, or to an intron-exon junction, or to anexon-intron junction listed with GenBank Accession No. NG_008466.1

Target site(s) useful in the practice of the invention are thoseinvolved in mRNA splicing (such as splice donor sites, splice acceptorsites or exonic splicing enhancer elements). Splicing branch points andexon recognition sequences or splice enhancers are also potential targetsites for modulation of mRNA splicing. In one embodiment,oligonucleotide compounds disclosed herein can bind to a selected targetnucleic acid sequence to induce exon skipping. In some embodiments,masking a donor splice site can induce exon skipping. In someembodiments, masking an acceptor splice site can induce exon skipping.For example, owing to the nature of morpholino oligomers, one ofordinary skill in the art can identify sequences that will reliably bindsplice junctions. As described in the examples herein, the efficacy oftargeted morpholino SSOs can be quickly ascertained in tissue culture.

Another modification of the oligonucleotide compounds disclosed hereininvolves chemically linking one or more moieties or conjugates to theoligonucleotide, which enhance the activity, cellular distribution, orcellular uptake of the oligonucleotide. These moieties or conjugates caninclude conjugate groups covalently bound to functional groups such asprimary or secondary hydroxyl groups. Non-limiting examples of moietiesand conjugates include lipid moieties (such as a cholesterol moiety, acholesteryl moiety, a thiocholesterol moeity), intercalators, reportermolecules, a palmityl moiety, or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety, a phospholipid, aliphaticchains (such as dodecandiol or undecyl residues), polyamine chains,polyamide chains, polyethylene glycol chains, polyether chains, cholicacid, and adamantane acetic acid. Examples of conjugate groups include,but are not limited to, cholesterols, lipids, phospholipids, biotin,phenazine, folate, phenanthridine, anthraquinone, acridine,fluoresceins, rhodamines, coumarins, and dyes.

Oligonucleotide compounds comprising lipophilic moieties, and methodsfor preparing such are known in the art, for example, as described inU.S. Pat. Nos. 5,138,045, 5,218,105 and 5,459,255, each of which isincorporated by reference in its entirety.

Representative United States patents that teach the preparation ofoligonucleotide compound conjugates include, but are not limited to,U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552, 538; 5,578,717; 5,580,731; 5,580,731; 5,591,584;5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486, 603; 5,512,439;5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762, 779;4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241; 5,391, 723; 5,416,203; 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599, 928 and 5,688,941, each of whichis hereby incorporated by reference in its entirety. Representativeconjugate groups are disclosed in U.S. Pat. Nos. 5,578,718; 6,153,737;6,287,860; and 6,783,931, each of which are incorporated by reference inits entirety.

The oligonucleotide compounds can be conveniently and routinely madethrough the established technique of solid phase synthesis. Equipmentuseful for such syntheses can be obtained through several commercialvendors, including Applied Biosystems (Foster City, Calif.). Synthesisof the oligonucleotide compounds is well understood by one of ordinaryskill in the art. It is also well known in the art to use similartechniques to prepare other oligonucleotides such as thephosphorothioates and alkylated derivatives. It is also well known touse similar techniques and commercially available modified amidites andcontrolled-pore glass (CPG) products such as biotin, fluorescein,acridine or psoralen-modified amidites and/or CPG (available from GlenResearch, Sterling Va.) to synthesize fluorescently labeled,biotinylated or other modified oligonucleotides such ascholesterol-modified oligonucleotides. Morpholinos, for example, arecommercially available through Gene Tools, LLC, Philomath Oreg.;http://www.gene-tools.com/). For example, the oligonucleotide compoundsof the invention (such as ASOs and SSOs) are synthesized in vitro and donot include antisense compositions of biological origin, or geneticvector constructs designed to direct the in vivo synthesis ofoligonucleotide compounds.

In one embodiment, the oligonucleotide compounds (e.g., modifiedoligonucleotide compounds) bind to coding and/or non-coding regions of atarget nucleic acid sequence of a subunit of the MMR system (such asMSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2), and modulate theexpression and/or function of the target molecule. In one embodiment,the oligonucleotide compounds (e.g., modified oligonucleotide compounds)bind to a natural antisense target nucleic acid sequence of a subunit ofthe MMR system (such as MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2),and modulate the expression and/or function of the target molecule. Inone embodiment, the oligonucleotide compounds bind to a sense targetnucleic acid sequence of a subunit of the MMR system (such as MSH2,MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2), and modulate the expressionand/or function of the target molecule.

Embodiments of the present invention are directed to oligonucleotidecompounds that hybridize with a complementary sequence of the human MSH2gene and/or mRNA. For example, mRNA may be pre-mRNA. The human MSH2 genecomprises SEQ ID NO: 35. Embodiments of the present invention can bedirected to modifying MSH2 gene expression. For example, embodiments ofthe present invention may be directed at skipping MSH2 exons.Embodiments of the present invention encompass splice switchingoligonucleotides (SSOs). In one embodiment, the oligonucleotide compoundcomprises at least one modification described herein. Embodiments of thepresent invention encompass morpholino oligonucleotides complementary tothe target nucleic acid sequence of MSH2.

Embodiments of the present invention are directed to oligonucleotidecompounds that hybridize with a complementary sequence of the human MSH3gene and/or mRNA. For example, mRNA may be pre-mRNA. The human MSH3 genecomprises SEQ ID NO: 36. Embodiments of the present invention can bedirected to modifying MSH3 gene expression. For example, embodiments ofthe present invention may be directed at skipping MSH3 exons.Embodiments of the present invention encompass splice switchingoligonucleotides (SSOs). In one embodiment, the oligonucleotide compoundcomprises at least one modification described herein. Embodiments of thepresent invention encompass morpholino oligonucleotides complementary tothe target nucleic acid sequence of MSH3.

Embodiments of the present invention are directed to oligonucleotidecompounds that hybridize with a complementary sequence of the human MSH6gene and/or mRNA. For example, mRNA may be pre-mRNA. The human MSH6 genecomprises SEQ ID NO: 37. Embodiments of the present invention can bedirected to modifying MSH6 gene expression. For example, embodiments ofthe present invention may be directed at skipping MSH6 exons.Embodiments of the present invention encompass splice switchingoligonucleotides (SSOs). In one embodiment, the oligonucleotide compoundcomprises at least one modification described herein. Embodiments of thepresent invention encompass morpholino oligonucleotides complementary tothe target nucleic acid sequence of MSH6.

Embodiments of the present invention are directed to oligonucleotidecompounds that hybridize with a complementary sequence of the human MLH1gene and/or mRNA. For example, mRNA may be pre-mRNA. The human MLH1 genecomprises SEQ ID NO: 38. Embodiments of the present invention can bedirected to modifying MLH1 gene expression. For example, embodiments ofthe present invention may be directed at skipping MLH1 exons.Embodiments of the present invention encompass splice switchingoligonucleotides (SSOs). In one embodiment, the oligonucleotide compoundcomprises at least one modification described herein. Embodiments of thepresent invention encompass morpholino oligonucleotides complementary tothe target nucleic acid sequence of MLH1.

Embodiments of the present invention are directed to oligonucleotidecompounds that hybridize with a complementary sequence of the human PMS1gene and/or mRNA. For example, mRNA may be pre-mRNA. The human PMS1 genecomprises SEQ ID NO: 39. Embodiments of the present invention can bedirected to modifying PMS1 gene expression. For example, embodiments ofthe present invention may be directed at skipping PMS1 exons.Embodiments of the present invention encompass splice switchingoligonucleotides (SSOs). In one embodiment, the oligonucleotide compoundcomprises at least one modification described herein. Embodiments of thepresent invention encompass morpholino oligonucleotides complementary tothe target nucleic acid sequence of PMS1.

Embodiments of the present invention are directed to oligonucleotidecompounds that hybridize with a complementary sequence of the human PMS2gene and/or mRNA. For example, mRNA may be pre-mRNA. The human PMS2 genecomprises SEQ ID NO: 40. Embodiments of the present invention can bedirected to modifying PMS2 gene expression. For example, embodiments ofthe present invention may be directed at skipping PMS2 exons.Embodiments of the present invention encompass splice switchingoligonucleotides (SSOs). In one embodiment, the oligonucleotide compoundcomprises at least one modification described herein. Embodiments of thepresent invention encompass morpholino oligonucleotides complementary tothe target nucleic acid sequence of PMS2.

Embodiments of the present invention encompass oligonucleotide compoundsthat hybridize with a complementary sequence of the human MLH3 geneand/or mRNA. For example, mRNA may be pre-mRNA. The human MLH3 genecomprises SEQ ID NO: 1. Embodiments of the present invention aredirected to modifying MLH3 gene expression. For example, embodiments ofthe present invention may be directed at skipping of MLH3 exon 7 (SEQ IDNO: 2). Embodiments of the present invention encompass splice switchingoligonucleotides (SSOs). Embodiments of the present invention includeoligonucleotide compounds comprising SEQ ID NO: 3 or SEQ ID NO: 4. Inone embodiment, the oligonucleotide compound comprises at least onemodification described herein. Embodiments of the present inventionencompass morpholino oligonucleotides complementary to the targetnucleic acid sequence of MLH3.

Target nucleic acid sequences of about 5-100 nucleotides in length,comprising a stretch of at least five (5) consecutive are suitable fortargeting. Target nucleic acid sequences can include DNA or RNAsequences that comprise at least 5 consecutive nucleotides from the5′-terminus of the gene encoding a subunit of the MMR system (such asMSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2). Target nucleic acidsequences can include DNA or RNA sequences that comprise at least 5consecutive nucleotides from the 3′-terminus of the gene encoding asubunit of the MMR system (such as MSH2, MSH3, MSH6, MLH1, MLH3, PMS1,or PMS2).

In one embodiment, the oligonucleotide compound binds to an antisensestrand of a particular target nucleic acid sequence (for example, asubunit of the MMR system (such as MSH2, MSH3, MSH6, MLH1, MLH3, PMS1,or PMS2)). The target nucleic acid sequences include coding as well asnon-coding regions. Generally, the oligonucleotide compound can be fromabout 10 nucleotides in length up to about 50 nucleotides in length. Inone embodiment, the oligonucleotide compounds of the invention are 10 to50 nucleotides in length. In one embodiment, the oligonucleotidecompounds are at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. Insome embodiments, the oligonucleotides are 15 nucleotides in length. Insome embodiments, the oligonucleotides are 20 nucleotides in length. Insome embodiments, the oligonucleotides are 25 nucleotides in length. Insome embodiments, the oligonucleotides are 20 nucleotides in length. Insome embodiments, the oligonucleotides are 30 nucleotides in length.

Kits, Diagnostics, and Therapeutics.

The oligonucleotide compounds of the present invention can be utilizedfor diagnostics, therapeutics, and prophylaxis, and as components ofkits. For example, the specificity and sensitivity of antisenseoligonucleotides can be harnessed for therapeutic uses. Oligonucleotidecompounds (such as antisense oligonucleotides disclosed herein) can beemployed as therapeutic moieties in the treatment of disease states insubjects, such as human subjects. For example, oligonucleotide compoundscan be useful therapeutics utilized in treatment regimens for treatmentof cells, tissues and animals, especially humans.

Transfer of an exogenous nucleic acid into a host cell or organism, suchas an oligonucleotide compound of the invention, can be assessed bydirectly detecting the presence of the nucleic acid in the cell ororganism. Detection can be achieved using several methods well known andpracticed in the art. For example, the presence of the exogenous nucleicacid can be detected by Southern blot or by a polymerase chain reaction(PCR) technique using primers that specifically amplify nucleotidesequences associated with the nucleic acid. Expression of the exogenousnucleic acids can also be measured using conventional methods includinggene expression analysis. For instance, mRNA produced from an exogenousnucleic acid (or its absence thereof) can be detected and quantifiedusing a Northern blot and reverse transcription PCR (RT-PCR).

Kits.

For use in kits and diagnostics and in various biological systems, theoligonucleotide compounds of the present invention, either alone or incombination with other compounds or therapeutics, are useful as tools indifferential and/or combinatorial analyses to elucidate expressionpatterns of a portion or the entire complement of genes expressed withincells and tissues, such as a subunit of the MMR system (e.g., MSH2,MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2).

The invention also provides kits for treatment of a subject with agenetic disease, wherein the kit comprises at least an oligonucleotidecompound, packaged in a suitable container, together with instructionsfor its use. In one embodiment, the invention provides for a kit for thetreatment of a DNA Repeat Expansion Disease (DRED), the kit comprisingan oligonucleotide compound discussed herein. In one embodiment, theinvention provides for a kit for the treatment of a DNA Repeat ExpansionDisease (DRED), the kit comprising at least two oligonucleotidecompounds discussed herein. In one embodiment, the DRED is any onelisted in Table 1. In one embodiment, the oligonucleotide compoundcomprises SEQ ID NO: 3 or SEQ ID NO: 4. In one embodiment, theoligonucleotide compound comprises a nucleic acid sequence depicted inTable 4. In one embodiment, the oligonucleotide compound comprises atleast one modification described herein. In some embodiments, the kitswill contain at least one oligonucleotide compound (e.g., an ASO orSSO), such as shown in Table 4, SEQ ID NO: 3 or SEQ ID NO: 4, or acocktail of antisense molecules comprising a combination of SEQ ID NO:3, SEQ ID NO:4, or a nucleic acid sequence depicted in Table 4. The kitscan also comprise any one, or a combination thereof, of the following:an oligonucleotide compound that is directed to a nucleic acid sequence(or target complementary nucleic acid sequence) corresponding to aregion of interest for any one of the exons 1-16 described herein forGenBank Accession No. NG_007110.2, or to an intron-exon junction, or toan exon-intron junction listed with GenBank Accession No. NG_007110.2;an oligonucleotide compound that is directed to a nucleic acid sequence(or target complementary nucleic acid sequence) corresponding to aregion of interest for any one of the exons 1-24 described herein forGenBank Accession No. NG_016607.1, or to an intron-exon junction, or toan exon-intron junction listed with GenBank Accession No. NG_016607.1;an oligonucleotide compound that is directed to a nucleic acid sequence(or target complementary nucleic acid sequence) corresponding to aregion of interest for any one of the exons 1-10 described herein forGenBank Accession No. NG_007111.1 or SEQ ID NO: 33, or to an intron-exonjunction, or to an exon-intron junction listed with GenBank AccessionNo. NG_007111.1 or SEQ ID NO: 33; an oligonucleotide compound that isdirected to a nucleic acid sequence (or target complementary nucleicacid sequence) corresponding to a region of interest for any one of theexons 1-19 described herein for GenBank Accession No. NG_007109.2, or toan intron-exon junction, or to an exon-intron junction listed withGenBank Accession No. NG_007109.2; an oligonucleotide compound that isdirected to a nucleic acid sequence (or target complementary nucleicacid sequence) corresponding to a region of interest for any one of theexons 1-13 described herein for GenBank Accession No. NG_008648.1, or toan intron-exon junction, or to an exon-intron junction listed withGenBank Accession No. NG_008648.1; an oligonucleotide compound that isdirected to a nucleic acid sequence (or target complementary nucleicacid sequence) corresponding to a region of interest for any one of theexons 1-13 described herein for GenBank Accession No. NG_008649.1 or SEQID NO: 1, or to an intron-exon junction, or to an exon-intron junctionlisted with GenBank Accession No. NG_008649.1 or SEQ ID NO: 1; or anoligonucleotide compound that is directed to a nucleic acid sequence (ortarget complementary nucleic acid sequence) corresponding to a region ofinterest for any one of the exons 1-15 described herein for GenBankAccession No. NG_008466.1, or to an intron-exon junction, or to anexon-intron junction listed with GenBank Accession No. NG_008466.1. Thekits may also contain peripheral reagents such as buffers, stabilizers,and the like.

The invention provides kits for monitoring the efficacy of treatment ina subject with a genetic disease, wherein the kit comprises at least oneprimer, packaged in a suitable container, together with instructions forits use. In one embodiment, the kit comprises at least two primers. Inone embodiment, the genetic disease is a DNA repeat expansion disease(DRED) listed in Table 1. In one embodiment, the status of a subunit ofthe MMR system (e.g., MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2) canbe monitored. In one embodiment, the primer comprises MLH3 L3324(TCCTTTCCTTCCGAGAGCTC, SEQ ID NO: 5). In one embodiment, the primercomprises MLH3×7L3138

(GCATTTCGATGTAGCCCTGG, SEQ ID NO: 29). In one embodiment, the primercomprises MLH3×7L3449 (TTGCCCGTTATCCAGAGGTT, SEQ ID NO: 30). In oneembodiment, the primer comprises MLH3 R3757 (TTTTCCGACCAGAGCCTTGT, SEQID NO: 6). In one embodiment, the primer comprises MLH3×7R3862(CAAGGCCCAGATCTTCCAGA, SEQ ID NO: 31). In one embodiment, the primercomprises MLH3×7R4013 (AGCTCCAGTTGTTCTCGGAT, SEQ ID NO: 32). The kitsmay also contain peripheral reagents such as buffers, stabilizers, andthe like.

The invention provides kits for monitoring the progression of a DNArepeat expansion disease (DRED), wherein the kit comprises at least oneprimer, packaged in a suitable container, together with instructions forits use. In one embodiment, the kit comprises at least two primers. Inone embodiment, the DRED is selected from the list in Table 1. In oneembodiment, the DRED is Fredreich Ataxia. In one embodiment, the statusof a subunit of the MMR system (e.g., MSH2, MSH3, MSH6, MLH1, MLH3,PMS1, or PMS2) can be monitored, indicative of the progression of arepeat expansion. In one embodiment, the MMR subunit is MLH3. In oneembodiment, the primer comprises MLH3 L3324 (TCCTTTCCTTCCGAGAGCTC, SEQID NO: 5). In one embodiment, the primer comprises MLH3×7L3138(GCATTTCGATGTAGCCCTGG, SEQ ID NO: 29). In one embodiment, the primercomprises MLH3×7L3449 (TTGCCCGTTATCCAGAGGTT, SEQ ID NO: 30). In oneembodiment, the primer comprises MLH3 R3757 (TTTTCCGACCAGAGCCTTGT, SEQID NO: 6). In one embodiment, the primer comprises MLH3×7R3862(CAAGGCCCAGATCTTCCAGA, SEQ ID NO: 31). In one embodiment, the primercomprises MLH3×7R4013 (AGCTCCAGTTGTTCTCGGAT, SEQ ID NO: 32). The kitsmay also contain peripheral reagents such as buffers, stabilizers, andthe like.

Treatments and Therapy for Diseases. As used herein and as is wellunderstood in the art, “treatment” is an approach for obtainingbeneficial or desired results, including clinical results. Beneficial ordesired clinical results can include, but are not limited to,alleviation or amelioration of one or more symptoms or conditions,diminution of extent of disease, a stabilized (i.e., not worsening)state of disease, preventing spread of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state andremission (whether partial or total), whether detectable orundetectable. “Treatment” can also refer to prolonging survival ascompared to expected survival if not receiving treatment.

The term “in need thereof” refers to the need for symptomatic orasymptomatic relief from a condition such as, for example, a DRED, acancer, a neurodegenerative disease, or a combination thereof. Thesubject in need thereof may or may not be undergoing treatment forconditions related to, for example, a DRED, a cancer, aneurodegenerative disease, or a combination thereof.

European patent application 13170245.8 claims an antisenseoligonucleotide directed against exon 43 of the dystrophin pre-mRNA,which facilitates the exclusion of exon 43 from the final mRNA. U.S.Pat. No. 8,361,979 claims antisense oligonucleotides that may be usedfor the treatment of Duchenne Muscular Dystrophy. U.S. Pat. No.8,455,634 claims an antisense molecule capable of binding to a selectedtarget site to induce exon skipping in the dystrophin gene. U.S.Application Publication No. US 2014/0039037 discloses claims directed toantisense oligonucleotides that induce skipping of exonic sequences thatcomprises the trinucleotide repeat expansion. These disclosures useantisense oligonucleotides to target the diseased gene itself, ratherthan disease modifying genes, such as a subunit of the MMR system (e.g.,MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2). A drawback of targetingthe diseased gene itself is that the proposed therapeutic has limitedeffectiveness for only a single disorder. For many of the expansiondisorders, such as FXS, DM1, DM2, and FRDA the repeat is in an intron,or in the first or last exon, which cannot be skipped. Presently, thereis no cure for any of the DNA repeat expansion disorders. Furthermore,the treatment options for these devastating disorders are generallyinadequate. In particular, there remains an urgent unmet need for newtherapeutic compositions that prevent the unwanted, progressivetrinucleotide repeat expansion associated with such disorders. Theobject of this present invention is to address this unmet need.

The present disclosure relates generally to small molecule therapeutics(e.g., oligonucleotide compounds, naked or modified) useful for thetreatment of DNA repeat expansion diseases (DREDs). In one embodiment,an oligonucleotide compound (e.g., an antisense oligonucleotide) isadministered to a subject to prevent or treat diseases or disordersassociated with DNA repeat expansion. In one embodiment, anoligonucleotide compound is directed to a target nucleic acid sequenceof a subunit of the MMR system (e.g., MSH2, MSH3, MSH6, MLH1, MLH3,PMS1, or PMS2). In one embodiment, an effective amount of theoligonucleotide compound is administered to the subject. In someembodiments, the oligonucleotide compound is a modified oligonucleotidethat is nuclease-resistant. In some embodiments, the oligonucleotidecompound comprises a pharmaceutical composition administered to asubject in a pharmaceutically acceptable carrier. In some embodiments,the oligonucleotide compound (e.g., an antisense oligonucleotide thatdirects exon skipping) can serve as a therapeutic method for thetreatment of various DREDs.

Embodiments of the invention may be used to treat human DRED. Inparticular, embodiments of the present invention may be used to treatdiseases associated with expanded DNA repeats. For example, expanded DNArepeat disorders may include trinucleotide repeat disorders. Examples ofdiseases which may benefit from embodiments of the present invention mayinclude Friedreich ataxia, ALS, Huntington's disease, Fragile Xsyndrome, Myotonic dystrophy Types I and II, Spino Cerebellar Ataxias(SCAs). SCAs may include SCA1, SCA2, SCA3, SCA6, SCAT, SCAB, SCA10 andSCA17. Additional examples of diseases, which may benefit fromembodiments of the present invention may include those disorders listedin Table 1. In one embodiment, the DRED is Duchenne Muscular Dystrophy,Fredreich Ataxia, or Huntington's disease. In some embodiments, the DREDis a disease selected from Table 1. Embodiments of the invention mayslow the rate of or inhibit repeat expansion. Embodiments of the presentinvention may slow the rate of or inhibit the progression of repeatexpansion disorders. Embodiments of the invention may slow the rate soas to inhibit the progression from an asymptomatic size to a diseasecausing size, thus preventing onset of an expansion disease.

For therapeutics, a subject, for example, a human, suspected of having adisease or disorder (such as a DRED), which can be treated by modulatingthe expression of a nucleic acid sequence of a subunit of the MMR system(e.g., MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2) is treated byadministering an oligonucleotide compound (such as an ASO) in accordancewith this invention. In one embodiment, a pharmaceutical compositioncomprising a an oligonucleotide compound disclosed herein, such as anuclease-resistant oligonucleotide 15 to 30 nucleotide bases in lengthtargeted to a complementary nucleic acid sequence of a gene or geneproduct encoding a MutS or MutL subunit, is administered to a subject.In one embodiment, the oligonucleotide hybridizes with and decreases theexpression of the human MutS or MutL subunit (such as MSH2, MSH3, MSH6,MLH1, MLH3, PMS1, or PMS2) by about 10%, about 20%, about 30%, about40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%,about 90%, about 95%, about 97%, about 98%, about 99%, or 100%, ascompared to a normal control. In one embodiment, the oligonucleotidecompound comprises at least one modification. In one embodiment, theoligonucleotide is 17 to 28 nucleotide bases in length. In oneembodiment, the oligonucleotide is 18 to 25 nucleotide bases in length.In one embodiment, the oligonucleotide is 19 to 23 nucleotide bases inlength.

In one embodiment, a pharmaceutical composition that is anoligonucleotide compound comprising an oligonucleotide complex can beadministered. In one embodiment, the complex comprises a firstoligonucleotide and a second oligonucleotide, wherein the firstoligonucleotide comprises a sequence complementary to an acceptor regionof an exon of a gene encoding a MutS or MutL subunit, and wherein thenucleic acid sequence of the first oligonucleotide comprises anuclease-resistant modification, and wherein the second oligonucleotidecomprises a sequence complementary to a donor region of an exon of agene encoding a MutS or MutL subunit, and wherein the nucleic acidsequence of the second oligonucleotide comprises a nuclease-resistantmodification. In another embodiment, the complex comprises a firstoligonucleotide and a second oligonucleotide, wherein the firstoligonucleotide comprises a sequence complementary to an acceptor regionof an exon of a gene encoding a MutS or MutL subunit, and wherein thenucleic acid sequence of the first oligonucleotide comprises anuclease-resistant modification, and wherein the second oligonucleotidecomprises a sequence complementary to a donor region of an exon of agene encoding a MutS or MutL subunit. In a further embodiment, thecomplex comprises a first oligonucleotide and a second oligonucleotide,wherein the first oligonucleotide comprises a sequence complementary toan acceptor region of an exon of a gene encoding a MutS or MutL subunit,and wherein the second oligonucleotide comprises a sequencecomplementary to a donor region of an exon of a gene encoding a MutS orMutL subunit, and wherein the nucleic acid sequence of the secondoligonucleotide comprises a nuclease-resistant modification. In oneembodiment, the human MutS or MutL subunit comprises MSH2, MSH3, MSH6,MLH1, MLH3, PMS1, or PMS2. In one embodiment, the methods comprise thestep of administering to the subject in need of treatment, atherapeutically effective amount of the oligonucleotide complex. Theoligonucleotide complex of the present invention effectively modulatesthe activity of a MutS or MutL subunit, or modulates the expression of aMutS or MutL subunit. In one embodiment, the activity or expression of aMutS or MutL subunit in an subject is decreased by about 10% as comparedto a control. In other embodiments, the activity or expression of a MutSor MutL subunit in a subject is decreased by about 20%. In yet otherembodiments, the activity or expression of a MutS or MutL subunit in asubject is decreased by about 30%. In some embodiments, the activity orexpression of a MutS or MutL subunit in a subject is decreased by about50%. In some embodiments, the activity or expression of a MutS or MutLsubunit in a subject is decreased by about 60%. In some embodiments, theactivity or expression of a MutS or MutL subunit in a subject isdecreased by about 70%. The oligonucleotide compounds disclosed herein(e.g., ASOs, SSOs, or oligonucleotide complexes) can modulate mRNAexpression of a MutS or MutL subunit (such as MSH2, MSH3, MSH6, MLH1,MLH3, PMS1, or PMS2) by at least 10%, by at least 20%, by at least 25%,by at least 30%, by at least 40%, by at least 50%, by at least 60%, byat least 70%, by at least 75%, by at least 80%, by at least 85%, by atleast 90%, by at least 95%, by at least 98%, by at least 99%, or by 100%as compared to a control. In one embodiment, the oligonucleotidecomprises SEQ ID NO: 3. In one embodiment, the oligonucleotide comprisesSEQ ID NO: 4. In one embodiment, the oligonucleotide comprises a nucleicacid sequence depicted in Table 4. In one embodiment, theoligonucleotide comprises an oligonucleotide compound that is directedto a nucleic acid sequence corresponding to a region of interest for anyone of the exons 1-16 described herein for GenBank Accession No.NG_007110.2, or to an intron-exon junction, or to an exon-intronjunction listed with GenBank Accession No. NG_007110.2. In oneembodiment, the oligonucleotide comprises an oligonucleotide compoundthat is directed to a nucleic acid sequence corresponding to a region ofinterest for any one of the exons 1-24 described herein for GenBankAccession No. NG_016607.1, or to an intron-exon junction, or to anexon-intron junction listed with GenBank Accession No. NG_016607.1. Inone embodiment, the oligonucleotide comprises an oligonucleotidecompound that is directed to a nucleic acid sequence corresponding to aregion of interest for any one of the exons 1-10 described herein forGenBank Accession No. NG_007111.1 or SEQ ID NO: 33, or to an intron-exonjunction, or to an exon-intron junction listed with GenBank AccessionNo. NG_007111.1 or SEQ ID NO: 33. In one embodiment, the oligonucleotidecomprises an oligonucleotide compound that is directed to a nucleic acidsequence corresponding to a region of interest for any one of the exons1-19 described herein for GenBank Accession No. NG_007109.2, or to anintron-exon junction, or to an exon-intron junction listed with GenBankAccession No. NG_007109.2. In one embodiment, the oligonucleotidecomprises an oligonucleotide compound that is directed to a nucleic acidsequence corresponding to a region of interest for any one of the exons1-13 described herein for GenBank Accession No. NG_008648.1, or to anintron-exon junction, or to an exon-intron junction listed with GenBankAccession No. NG_008648.1. In one embodiment, the oligonucleotidecomprises an oligonucleotide compound that is directed to a nucleic acidsequence corresponding to a region of interest for any one of the exons1-13 described herein for GenBank Accession No. NG_008649.1 or SEQ IDNO: 1, or to an intron-exon junction, or to an exon-intron junctionlisted with GenBank Accession No. NG_008649.1 or SEQ ID NO: 1. In oneembodiment, the oligonucleotide comprises an oligonucleotide compoundthat is directed to a nucleic acid sequence corresponding to a region ofinterest for any one of the exons 1-15 described herein for GenBankAccession No. NG_008466.1, or to an intron-exon junction, or to anexon-intron junction listed with GenBank Accession No. NG_008466.1.

For example, the decrease or reduction of the expression of a MutS orMutL subunit (such as MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2) canbe measured in serum, blood, adipose tissue, cerebral spinal fluid,liver, or any other body fluid, tissue or organ of the subject. In oneembodiment, the cells contained within the above-listed fluids, tissuesor organs that are being analyzed contain a nucleic acid moleculeencoding a MutS or MutL subunit (such as MSH2, MSH3, MSH6, MLH1, MLH3,PMS1, or PMS2).

Formulations and Administration

The oligonucleotide compounds of the invention can be utilized inpharmaceutical compositions by adding an effective amount of a compoundto a suitable pharmaceutically acceptable diluent or carrier. Use of theoligonucleotide compounds and methods of the invention may also beuseful prophylactically.

An “effective amount”, “sufficient amount” or “therapeutically effectiveamount” as used herein is an amount of a composition that is sufficientto effect beneficial or desired results, including clinical results. Assuch, the effective amount may be sufficient, for example, to reduce orameliorate the severity and/or duration of an affliction or condition,or one or more symptoms thereof, prevent the advancement of conditionsrelated to an affliction or condition, prevent the recurrence,development, or onset of one or more symptoms associated with anaffliction or condition, or enhance or otherwise improve theprophylactic or therapeutic effect(s) of another therapy. An effectiveamount also includes the amount of the composition (e.g., theoligonucleotide compounds discussed herein) that avoids or substantiallyattenuates undesirable side effects.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which a compound is administered. Non-limiting examples of suchpharmaceutical carriers include liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.The pharmaceutical carriers may also be saline, gum acacia, gelatin,starch paste, talc, keratin, colloidal silica, urea, and the like. Inaddition, auxiliary, stabilizing, thickening, lubricating and coloringagents may be used. Other examples of suitable pharmaceutical carriersare described in Remington: The Science and Practice of Pharmacy,21^(st) Edition (University of the Sciences in Philadelphia, ed.,Lippincott Williams & Wilkins 2005); and Handbook of PharmaceuticalExcipients, 7^(th) Edition (Raymond Rowe et al., ed., PharmaceuticalPress 2012); each hereby incorporated by reference in its entirety.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto. Apharmaceutically acceptable carrier can comprise any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. The use of such media and agents forpharmaceutically active substances is well known in the art. Anyconventional media or agent that is compatible with the active compoundcan be used. Supplementary active compounds can also be incorporatedinto the compositions. For oligonucleotide compounds, examples ofpharmaceutically acceptable salts and their uses are further describedin U.S. Pat. No. 6,287,860, which is hereby incorporated by reference inits entirety.

In one embodiment, modulation of a subunit of the MMR system (e.g.,MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2) can be effected byadministering one or more oligonucleotide compounds (e.g., ASOs or SSOs,naked or modified) to a subject in need thereof. In one embodiment, theprevention, amelioration, or treatment of a DRED that is related toabnormal expression, function, activity of a subunit of the MMR system(e.g., MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, or PMS2) as compared to anormal control can also be effected by administering one or moreoligonucleotide compounds (e.g., ASOs or SSOs, naked or modified) to asubject in need thereof. Embodiments of the present invention can beadministered alone, or can be administered in a therapeutic cocktail oras a pharmaceutical composition. For example, a pharmaceuticalcomposition can comprise embodiments of the present invention, and asaline solution that includes a phosphate buffer. Embodiments of thepresent invention can be administered using the means and dosesdescribed herein. Embodiments of the present invention can beadministered in combination with a suitable carrier. In one embodiment,the oligonucleotide compounds of the invention (e.g., ASOs and SSOs)encompass any pharmaceutically acceptable salts, esters, or salts ofsuch esters, or any other compound which, upon administration to asubject, provides (directly or indirectly) the biologically activemetabolite or residue thereof.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEM™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, a pharmaceutically acceptable polyol like glycerol,propylene glycol, liquid polyethylene glycol, and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, itcan be useful to include isotonic agents, for example, sugars,polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating thecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated herein, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated herein. In the case of sterile powders for the preparation ofsterile injectable solutions, examples of useful preparation methods arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed.

Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orsterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The oligonucleotide compounds of the invention may also be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Non-limiting examples of United States patents that teach thepreparation of such uptake, distribution and/or absorption-assistingformulations include U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,165; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595.756, each of which is herein incorporated byreference.

For treating tissues in the central nervous system, administration canbe made by, e.g., injection or infusion into the cerebrospinal fluid.Administration of antisense RNA into cerebrospinal fluid is described,e.g., in U.S. Pat. No. 7,622,455, which is incorporated by reference inits entirety. When it is intended that an oligonucleotide compound(e.g., an ASO or SSO) will be administered to cells of the centralnervous system, administration can be with one or more agents capable ofpromoting penetration of the oligonucleotide compound across theblood-brain barrier. Injection can be made, e.g., in the entorhinalcortex or hippocampus. See also U.S. Pat. Nos. 6,632,427 and 6,756,523for additional disclosures relating to direct delivery to the brain,each patent which is incorporated by reference in its entirety. Fortreating cardiac tissues, administration can be made by, e.g., injectionor infusion into the bloodstream. The injection can be administered bythe following routes: intraperitoneal injection, subcutaneous injection,intradermal injection, intravenous injection, intramuscular injection,intra-arterial injection, or a combination thereof. In one embodiment,administration into the bloodstream is useful to treat the heart, whichis a major affected target in Friedreich ataxia.

Formulations useful for topical administration include those in whichthe oligonucleotide compounds of the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Exemplarylipids and liposomes include neutral (e.g. diolcoyl-phosphatidylethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC),distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidylglycerol (DMPG)) and cationic (e.g. diolcoyltetramethyl-aminopropyl(DOTAP), and diolcoyl-phosphatidyl ethanolamine (DOTMA)). For topical orother administration, oligonucleotide compounds of the invention can beencapsulated within liposomes or can form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotidecompounds can be complexed to lipids, in particular to cationic lipids.Exemplary fatty acids and esters, pharmaceutically acceptable saltsthereof, and their uses are further described in U.S. Pat. No.6,287,860, which is hereby incorporated by reference in its entirety.

The formulation of therapeutic compositions and their subsequentadministration (dosing) is believed to be within the skill of those inthe art. Dosing is dependent on severity and responsiveness of thedisease state to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved. Optimal dosing schedulescan be calculated from measurements of drug accumulation in the body ofthe patient. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Optimum dosages mayvary depending on the relative potency of individual oligonucleotides,and can generally be estimated based on EC₅₀s found to be effective inin vitro and in vivo animal models. In some embodiments, thetherapeutically effective amount is at least about 0.1 mg/kg bodyweight, at least about 0.25 mg/kg body weight, at least about 0.5 mg/kgbody weight, at least about 0.75 mg/kg body weight, at least about 1mg/kg body weight, at least about 2 mg/kg body weight, at least about 3mg/kg body weight, at least about 4 mg/kg body weight, at least about 5mg/kg body weight, at least about 6 mg/kg body weight, at least about 7mg/kg body weight, at least about 8 mg/kg body weight, at least about 9mg/kg body weight, at least about 10 mg/kg body weight, at least about15 mg/kg body weight, at least about 20 mg/kg body weight, at leastabout 25 mg/kg body weight, at least about 30 mg/kg body weight, atleast about 40 mg/kg body weight, at least about 50 mg/kg body weight,at least about 75 mg/kg body weight, at least about 100 mg/kg bodyweight, at least about 200 mg/kg body weight, at least about 250 mg/kgbody weight, at least about 300 mg/kg body weight, at least about 350mg/kg body weight, at least about 400 mg/kg body weight, at least about450 mg/kg body weight, or at least about 500 mg/kg body weight.

In one embodiment, the oligonucleotide compound can be administered tothe subject one time (e.g., as a single injection or deposition).Alternatively, administration can be once or twice daily to a subject inneed thereof for a period of from about 2 to about 28 days, or fromabout 7 to about 10 days, or from about 7 to about 15 days. It can alsobe administered once or twice daily to a subject for a period of 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times per year, or a combinationthereof. For example, the dosage may be given once or more daily,weekly, monthly or yearly, or even once every 2 to 20 years. In oneembodiment, two or more combined oligonucleotide compounds,therapeutics, and the like may be used together in combination orsequentially. The dosage can vary depending upon known factors such asthe pharmacodynamic characteristics of the active ingredient and itsmode and route of administration; time of administration of activeingredient; age, sex, health and weight of the recipient; nature andextent of symptoms; kind of concurrent treatment, frequency of treatmentand the effect desired; and rate of excretion. Persons of ordinary skillin the art can easily estimate repetition rates for dosing based onmeasured residence times and concentrations of the drug in bodily fluidsor tissues. Following successful treatment, it may be desirable to havethe patient undergo maintenance therapy to prevent the recurrence of thedisease state, wherein the oligonucleotide compound is administered inmaintenance doses, ranging from at least about 0.1 mg/kg body weight toabout 10 mg/kg of body weight, once or more daily, to once every 2-20years. Certain injected dosages of antisense oligonucleotides, forexample, are described, in U.S. Pat. No. 7,563,884, which is herebyincorporated by reference in its entirety.

While the embodiments of the present invention are described withreference to various implementations and exploitations, it will beunderstood that these embodiments are illustrative and that the scope ofthe inventions is not limited to them. Many variations, modifications,additions, and improvements are possible. Further still, any stepsdescribed herein may be carried out in any desired order, and anydesired steps may be added or deleted. Support for the present inventionand additional embodiments of the present invention may be found in theattached documents all of which are expressly incorporated herein intheir entirety by reference hereto. Also, the phraseology andterminology used herein is for the purpose of description and should notbe regarded as limiting. The use of “including,” “comprising,” or“having,” “containing,” “involving,” and variations thereof herein, ismeant to encompass the items listed thereafter and equivalents thereofas well as additional items.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Exemplary methods and materialsare described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ortesting of the present invention.

As will be apparent to one of ordinary skill in the art from a readingof this disclosure, the embodiments of the present disclosure can beembodied in forms other than those specifically disclosed above. Theparticular embodiments described herein are, therefore, to be consideredas illustrative and not restrictive. Those skilled in the art willrecognize, or be able to ascertain, using no more than routineexperimentation, numerous equivalents to the specific embodimentsdescribed herein. The scope of the invention is as set forth in theappended claims and equivalents thereof, rather than being limited tothe examples contained in the foregoing description.

All publications and other references mentioned herein are incorporatedby reference in their entirety, as if each individual publication orreference were specifically and individually indicated to beincorporated by reference. Publications and references cited herein arenot admitted to be prior art.

EXAMPLES

Examples are provided below to facilitate a more complete understandingof the invention. The following examples illustrate the exemplary modesof making and practicing the invention. However, the scope of theinvention is not limited to specific embodiments disclosed in theseExamples, which are for purposes of illustration only, since alternativemethods can be utilized to obtain similar results.

Example 1 Friedreich Ataxia is Characterized by Progressive RepeatExpansion

Friedreich ataxia (FRDA) is a progressive neurodegenerative disordercaused by GAA⋅TTC repeat expansion in the first intron of the frataxin(FXN) gene (3). Disease severity correlates to the length of theexpanded repeats and the consequent reduction of FXN gene expression.While the mechanism of repeat expansion is not yet understood, we havedeveloped versatile human cell models with an integrated “tandemreporter” (15) that recapitulate the expansion seen in FRDA patientsthat have allowed us to make rapid progress in understanding theexpansion process.

This model has a key advantage over authentic FXN repeat expansion: therepeats are not linked to an essential gene. Thus, the well-knownproblems associated with selection against frataxin knockdown cells inculture are able to be avoided (16). Further advantages of this systeminclude a single copy genomic location, the ability to control thetranscription into a repeat, and a cell environment permissive forexpansion. The “tandem reporter” expansion system uses modified HEK293cells. These cells express a large number of proteins typicallyexpressed exclusively, or preferentially, in neurons probably due topreferential transformation of neuronal lineage by adenovirus 5 inembryonic kidney (17,18). The ability to study expansion with anaccelerated time course is aided by the neuronal nature of HEK293 cells.Long uninterrupted GAA⋅TTC repeats cannot be propagated in bacteria.Therefore repeat arrays are built for the constructs using an in vitroligation strategy devised (19) circumventing bacteria. This gives thesystem another advantage since defined, uninterrupted repeats are used.

In models, the repeats expand incrementally, continuously and nearlysynchronously. Importantly, the rate of expansion is linked to the levelof transcription into the repeats (20). Thus, some therapeuticstrategies aimed at increasing transcription of the FXN gene willinadvertently increase repeat expansion.

These models have been used to determine that MSH3 (MutSbeta) isrequired for GAA⋅TTC repeat expansion (21). MutSbeta is comprised ofMSH2 and MSH3, and binds to a site targeted for mismatch repair.Multiple lines of evidence highlight the importance of MutSbeta inrepeat expansion: (i) shRNA knockdown of either MSH2 or MSH3 slowedGAA⋅TTC expansion in human cells, and (ii) ectopic expression ofMutSbeta induced GAA⋅TTC repeat expansion in the native FXN gene. Onceit binds to DNA to initiate repeat expansion, MutSbeta recruits a MutLcomplex.

The models have also been used to determine that MLH1 complexed withMLH3 (MutLgamma) is the next step required for GAA⋅TTC repeat expansion.Furthermore, MLH3 is involved in repeat expansion and only one of thetwo MLH3 alternative splicing isoforms is required.

Human MLH3 has 2 Isoforms

MLH3 is expressed in humans as two isoforms, MLH3iso1 and MLH3iso2,resulting from alternative splicing. MLH3iso1 includes exon 7, whichcontains a highly conserved portion of an endonuclease domain, whileMLH3 isoform 2 lacks this 72 base exon (FIG. 9B). Primer pairs, MLH3L3324 and MLH3 R3757, were used to detect MLH3iso1 and MLH3iso2 (FIG.9A). This pair resulted in a 434 bp band for MLH3iso1 and a 362 bp bandfor MLH3iso2; a 16.6% difference allowing easy visualization of thepresence or absence of the 72 nucleotide long exon 7. Dilutions ofdefined isoform templates containing or excluding exon 7 were done in10:1, 1:1, and 1:10 respectively to demonstrate the quantitative natureof this PCR.

Forcing MLH3 Exon Choice

MLH3 expression is key to GAA⋅TTC expansion in human cells. Manipulationof this minor component of MMR in a repeat expansion model may be apossible therapeutic target to limit DNA repeat expansion in FRDApatients. As MLH3iso2 lacks exon 7, which contains part of itsendonuclease domain, forced expression of this isoform may serve such apurpose. Splice switching oligos (SSOs) were designed to mask theacceptor and donor regions of MLH3 exon 7, inducing skipping of exon 7and the consequent production of MLH3iso2 (FIG. 5). Forcing exclusion ofexon 7 may approximate a functional knockout of the endonucleaseactivity of MLH3, which is critical for repeat expansion. Skipping exon7 leaves MLH3 isoform 2 intact, so will not impact the total cellularratios of MLH1 and its binding partners PMS2, PMS1 and MLH3. ExemplarySSOs are depicted in SEQ ID NOS: 3 and 4 below:

ML3X7acceptor6 (SEQ ID NO: 3) 5′-TCCCACctagatgagcaaggattgt-3′ML3X7donor8 (SEQ ID NO: 4) 5′-tctggctgcaaacagatccttacCA-3′

Small Molecule Directed Skipping of MLH3 Exon 7 Slows GAA⋅TTC RepeatExpansion

These cells were treated with splice switching oligonucleotides designedto exclude exon 7 of MLH3, so that the cells would preferentially makeMLH3iso2. Specifically, acceptor, donor or both SSOs were given twice aweek to FRDA model cells in culture. After 3 weeks in culture withvarious treatments, RT-PCR was used to measure the relative expressionof MLH3iso1 and MLH3iso2, and PCR on genomic DNA was used to measure thelength of the GAA⋅TTC repeat. RT-PCR demonstrates that the combinationof acceptor and donor SSOs at 500 nM most effectively excluded exon 7(FIG. 6). Correlated with the preferential expression of MLH3iso2, PCRanalysis of GAA⋅TTC expansion shows a reduced expansion rate (FIG. 7).

From these experiments, it is concluded that (i) MLH3 contributes toGAA⋅TTC repeat expansion in human cells, (ii) the endonuclease domain ofMLH3 is needed for this effect, and (iii) a small molecule therapeuticdirected at skipping of MLH3 exon 7 may be therapeutic avenue to slowthe progression of repeat expansion disorders such as Friedreich ataxia.

REFERENCES

-   1. Gatchel, J. R. and Zoghbi, H. Y. (2005) Diseases of unstable    repeat expansion: mechanisms and common principles. Nature reviews.    Genetics, 6, 743-755.-   2. Mirkin, S. M. (2007) Expandable DNA repeats and human disease.    Nature, 447, 932-940.-   3. Campuzano, V., Montermini, L., Molto, M. D., Pianese, L., Cossee,    M., Cavalcanti, F., Monros, E., Rodius, F., Duclos, F.,    Monticelli, A. et al. (1996) Friedreich's ataxia: autosomal    recessive disease caused by an intronic GAA triplet repeat    expansion. Science, 271, 1423-1427.-   4. Chauhan, C., Dash, D., Grover, D., Rajamani, J. and    Mukerji, M. (2002) Origin and instability of GAA repeats: insights    from Alu elements. J Biomol Struct Dyn, 20, 253-263.-   5. Clark, R. M., Dalgliesh, G. L., Endres, D., Gomez, M., Taylor, J.    and Bidichandani, S. I. (2004) Expansion of GAA triplet repeats in    the human genome: unique origin of the FRDA mutation at the center    of an Alu. Genomics, 83, 373-383.-   6. Batzer, M. A. and Deininger, P. L. (2002) Alu repeats and human    genomic diversity. Nature reviews. Genetics, 3, 370-379.-   7. Matsuura, T., Fang, P., Lin, X., Khajavi, M., Tsuji, K.,    Rasmussen, A., Grewal, R. P., Achari, M., Alonso, M. E.,    Pulst, S. M. et al. (2004) Somatic and germline instability of the    ATTCT repeat in spinocerebellar ataxia type 10. American journal of    human genetics, 74, 1216-1224.-   8. Kurosaki, T., Ueda, S., Ishida, T., Abe, K., Ohno, K. and    Matsuura, T. (2012) The unstable CCTG repeat responsible for    myotonic dystrophy type 2 originates from an AluSx element insertion    into an early primate genome. PloS one, 7, e38379.-   9. Prolla, T. A., Pang, Q., Alani, E., Kolodner, R. D. and    Liskay, R. M. (1994) MLH1, PMS1, and MSH2 interactions during the    initiation of DNA mismatch repair in yeast. Science, 265, 1091-1093.-   10. Flores-Rozas, H. and Kolodner, R. D. (1998) The Saccharomyces    cerevisiae MLH3 gene functions in MSH3-dependent suppression of    frameshift mutations. Proceedings of the National Academy of    Sciences of the United States of America, 95, 12404-12409.-   11. Raschle, M., Marra, G., Nystrom-Lahti, M., Schar, P. and    Jiricny, J. (1999) Identification of hMutLbeta, a heterodimer of    hMLH1 and hPMS1. The Journal of biological chemistry, 274,    32368-32375.-   12. Lipkin, S. M., Wang, V., Jacoby, R., Banerjee-Basu, S.,    Baxevanis, A D., Lynch, H. T., Elliott, R. M. and    Collins, F. S. (2000) MLH3: a DNA mismatch repair gene associated    with mammalian microsatellite instability. Nature genetics, 24,    27-35.-   13. Tian, L., Hou, C., Tian, K., Holcomb, N. C., Gu, L. and    Li, G. M. (2009) Mismatch recognition protein MutSbeta does not    hijack (CAG)n hairpin repair in vitro. The Journal of biological    chemistry, 284, 20452-20456.-   14. Cannavo, E., Marra, G., Sabates-Bellver, J., Menigatti, M.,    Lipkin, S. M., Fischer, F., Cejka, P. and Jiricny, J. (2005)    Expression of the MutL homologue hMLH3 in human cells and its role    in DNA mismatch repair. Cancer Res, 65, 10759-10766.-   15. Banerjee, A., Sammarco, M. C., Ditch, S., Wang, J. and    Grabczyk, E. (2009) A novel tandem reporter quantifies RNA    polymerase II termination in mammalian cells. PloS one, 4, e6193.-   16. Calmels, N., Seznec, H., Villa, P., Reutenauer, L., Hibert, M.,    Haiech, J., Rustin, P., Koenig, M. and Puccio, H. (2009) Limitations    in a frataxin knockdown cell model for Friedreich ataxia in a    high-throughput drug screen. BMC Neurol, 9, 46.-   17. Graham, F. L., Smiley, J., Russell, W. C. and Nairn, R. (1977)    Characteristics of a human cell line transformed by DNA from human    adenovirus type 5. The Journal of general virology, 36, 59-74.-   18. Shaw, G., Morse, S., Ararat, M. and Graham, F. L. (2002)    Preferential transformation of human neuronal cells by human    adenoviruses and the origin of HEK 293 cells. The FASEB journal:    official publication of the Federation of American Societies for    Experimental Biology, 16, 869-871.-   19. Grabczyk, E. and Usdin, K. (1999) Generation of microgram    quantities of trinucleotide repeat tracts of defined length,    interspersion pattern, and orientation. Analytical biochemistry,    267, 241-243.-   20. Ditch, S., Sammarco, M. C., Banerjee, A. and Grabczyk, E. (2009)    Progressive GAA⋅TTC repeat expansion in human cell lines. PLoS    genetics, 5, e1000704.-   21. Halabi, A., Ditch, S., Wang, J. and Grabczyk, E. (2012) DNA    mismatch repair complex MutSbeta promotes GAA.TTC repeat expansion    in human cells. The Journal of biological chemistry, 287,    29958-29967.

Example 2

MLH3 expression is key to GAA⋅TTC expansion in human cells and forms thebasis for the first therapeutic to slow the expansion rate in Friedreichataxia and perhaps other repeat expansion diseases. This minor componentof mismatch repair (MMR) will be developed as a therapeutic target tolimit repeat expansion in FRDA patients. in vivo efficacy and safetystudies will be conducted in a mouse model. Without being bound bytheory, selective expansion of GAA⋅TTC repeats in disease relevanttissues to a critical size drives disease onset and progression in FRDA.Somatic expansion of GAA⋅TTC repeats requires transcription through therepeat then the sequential actions of MutSβ (MSH2/MSH3 heterodimer) andMutLγ (MLH1/MLH3 heterodimer). MSH3 expression was linked to activeGAA⋅TTC expansion in FRDA patient primary cells (G1). Similarly, MMR orMSH3 expression has been linked to region specific expansion of CAG⋅CTGrepeats in the Huntington's disease (HD) “R6” mouse model (G2-G4). MLH3operates downstream of MSH3. Without being bound by theory, MLH3, whilea minor player in canonical MMR, is a major force in DNA repeatexpansion. MLH3 has also recently been identified as a component ofCAG⋅CTG expansion in the HD “R6” mouse (G5).

Like the HD “R6” mouse, the FRDA “YG22” mouse model exhibits regionspecific GAA⋅TTC repeat expansion (G6). Splice-switchingoligonucleotides (SSOs) will be used to block this expansion as a firststep leading to human trials. The SSOs will be targeted to the mismatchrepair protein MLH3. MLH3 is expressed in humans as two isoforms, MLH3isoform 1 and MLH3 isoform 2, due to alternative splicing. MLH3 isoform1 includes exon 7, which contains a conserved endonuclease domain, whileMLH3 isoform 2 lacks exon 7. It has been recently found that MLH3isoform 1 is required for GAA⋅TTC expansion, while isoform 2 is not.Skipping exon 7 by use of SSOs effectively shifts MLH3 to isoform 2 andstops repeat expansion in human cells. Finally, skipping exon 7 leavesMLH3 isoform 2 intact, the total cellular ratios of MLH1 and its bindingpartners PMS2, PMS1 and MLH3 will not be impacted. The mouse MLH3 exonstructure parallels that of humans.

This approach targets a central mechanism that is likely shared by allrepeat expansion diseases. Therefore, it has the potential to treatmany, if not all of the diseases in this class. The SSOs to be used arethe same type already in human trials for Duchene muscular dystrophy(G7-G9). Consequently, this project has great translational potential.

MMR has been implicated in repeat expansions of numerous disordersincluding Huntington's disease (HD) and myotonic dystrophy (DM)(G10-G13). Although somatic mosaicism of GAA⋅TTC allele size in FRDApatients has long been known (G14-G16) consideration of a role for MMRin the underlying GAA⋅TTC repeat expansion is more recent (G1,G17-G19).

In the MMR pathway, MutS heterodimers are responsible for identifyingand binding mismatched bases and/or insertion/deletion loops of varyingsize (G20). MSH2 (MutS Homologue 2) is a component of both MutScomplexes and has consequently been implicated in DNA repeat expansion(G10, G17, G19). Upon mismatch recognition by a MutS complex, a MutLheterodimer is recruited to make an incision near the lesion recognitionsite (G21-G23). Under physiologic conditions, binding of MutL initiatesrecruitment of the necessary machinery that will excise the lesion andsynthesize the DNA patch.

While the mechanism of repeat expansion is not yet fully understood,that GAA⋅TTC expansion rate is associated with transcription within therepeat (FIGS. 1A-F) and requires the action of mismatch repair (MMR)complex MutSb rather than MutSa (G1, G24). Much of the accumulatedevidence agrees that the MutSb complex, and/or the MSH3 subunit inparticular, is rate limiting for expansion seen in Huntington's disease(HD), myotonic dystrophy (DM) (G12, G13, G25, G26) as well as in FRDA(G1).

Analogous to the role of MSH2 in MutS complexes, MLH1 (MutL Homologue 1)is the core subunit of known MutL complexes. MLH1 combines with one ofthree partners called PMS1 (post-meiotic segregation increased 1), PMS2,and MLH3 to form MutLb, MutLa, and MutLg respectively. It is estimatedthat about 90% of MLH1 in most human cells is bound to PMS2 (MutLa)(G28, G29); further, PMS1 and PMS2 are estimated to be present in 10fold and 60 fold molar excess of MLH3 (G29). MutLα and MutLg appear tohave a role in MMR while MutLb does not. As with MSH2, MLH1 depletion isstrongly associated with hereditary nonpolyposis colorectal cancer(HNPCC) and sporadic gastric and endometrial carcinomas (G30-G32); PMS2depletion is also associated with HNPPC albeit to a lesser extent thanMLH1. Currently, evidence for MLH3 indicates that it rarely, if ever,contributes to cancer development (G33-G35).

PMS1, PMS2, and MLH3 have all been reported to compete for the samebinding site on the C-terminal of MLH1 (G36); as with the MutShomologues, it is possible that the abundance of these proteins inrelation to one another regulates their ability to compete for MLH1 andhence, their stability. Expression of MLH1 and PMS2 correlate strongly;however, evidence for MLH3 implicates that in addition to its alreadylow abundance, MLH3 expression may not be tethered to the expression ofother MutL proteins (G29). Generally, MLH3 is better understood for itsrole in meiotic recombination and the repair of frame-shift mutationsthan in canonical MMR (G37, G38). Interactions between MLH3, MLH1, andMSH3 have all been reported (G36-G38).

In the current working model shown in FIGS. 1A-F, resolution of astructure formed by transcription causes an out-of-register re-annealingof the two strands that leads to loop-outs. Without being bound bytheory, the MMR pathway is aberrantly activated by these small loop-outsin the repeat. The contribution of MutL subunits to GAA⋅TTC repeatexpansion will be assessed in the human cellular model of FRDA.Expression of MLH1, PMS2, and MLH3 was depleted in the cellular modeland changes in expansion rate over time were quantified.

MLH3 Expression is Key to GAA⋅TTC Expansion in Human Cells.

Lentiviral mediated shRNA knockdown of MLH1, PMS2 and MLH3 was carriedout in four independent clones of HEK293 cells carrying a single copy ofthe tandem reporter vector bearing 176 GAA⋅TTC repeats (G24). Eachknockdown used a pool of four shRNA-expressing lentivirus. After 4 weeksin culture, DNA and protein extracts were prepared as describedpreviously (G1). A representative PCR sizing gel is shown in FIG. 2A.DNA from MLH1 knockdown cells (MLH1sh) can be seen to have reducedexpansion compared to the empty vector control cells (pLKO) at week 4.DNA from MLH3 knockdown cells exhibit the least expansion (MLH3sh). Thenumber of triplet repeats gained in 4 weeks was calculated for all fourcell lines and presented in graphical form in FIG. 2B. MLH1 knockdownand MLH3 knockdown each showed a substantial and statisticallysignificant reduction in expansion rate. In contrast, PMS2 knockdownsamples showed a trend towards greater expansion. Thus shRNA knockdownof MMR proteins MLH1 and MLH3, but not PMS2 slows GAA⋅TTC expansion inhuman cells, indicating a role for MutLg

Like MutS, MutL partners are more stable as heterodimers (G28). Westernblot analysis of protein extracts from the cells showed the expectedreduction of both PMS2 and MLH1 in the MLH1 knockdown cells as comparedto controls (FIG. 3 compare lanes MLH1sh and lanes C). When PMS2 wasknocked down, the protein level of MLH1 decreased, but was still evident(FIG. 3 lanes PMS2sh). In contrast with MLH1 and PMS2, MLH3 knockdownled to a slight decrease in MLH1 protein but PMS2 protein did notchange. Because PMS2 stability depends on binding to MLH1, the lack ofchange in PMS2 with MLH3 knockdown indicates that MutLα levels areunaffected by the loss of MLH3 in these cells.

Taken together these data indicate that the necessary complex forGAA⋅TTC expansion is MutLg a heterodimer of MLH1 and MLH3. The lack ofconnection between MLH3 and cancer (G33-G35), and that reducing MLH3levels did not affect levels of MutSa, which is linked to cancer(G30-G32), suggested MLH3 as a possible therapeutic target to limit DNArepeat expansion in FRDA patients.

MLH3 Exon Skipping as an Alternative to MLH3 Knockdown.

MLH3 is expressed in humans as two isoforms (FIG. 4). MLH3 isoform 1includes exon 7, which contains a highly conserved portion of anendonuclease domain, while MLH3 isoform 2 lacks this 72 base exon (FIG.4). Without being bound by theory, if the endonuclease activity of MLH3is critical to repeat expansion, then exclusion of exon 7 would stoprepeat expansion. Skipping exon 7 leaves MLH3 isoform 2 intact, andtherefore does not impact the cellular ratios of MLH1 and its bindingpartners PMS2, PMS1 and MLH3.

MLH3 exon 7 was skipped using oligonucleotide analogues that would bindand mask the splice donor and acceptor signals flanking exon 7 in theunspliced pre-mRNA (FIG. 5). DNA oligonucleotides were also designed toassay the ratio of MLH3 isoform 1 and MLH3 isoform 2 mRNA via reversetranscription PCR (RT-PCR) in order to quantify the efficacy of theSSOs.

Morpholinos are oligonucleotide analogues that bind their complementarytarget DNA or RNA very tightly due to their uncharged backbone ofmorpholino subunits, which also makes them resistant to nucleases andproteases (G39). The SSOs used in tissue culture are “vivo-morpholinos”that have an octaguanidine moiety conjugated to the morpholino toenhance cellular uptake (G40).

SSOs Effectively Changed MLH3 from Isoform 1 to Isoform 2 and ReducedRepeat Expansion.

Different concentrations of acceptor SSO, donor SSO and combinations ofthe two were examined. An example of the experiments conducted with theSSOs is shown in FIG. 6. The acceptor SSO gave a graded concentrationdependent effect on exon skipping and the donor SSO was somewhat moreeffective, showing a steeper gradient. However, efficacy of spliceswitching was greatly enhanced when donor and acceptor SSOs were used incombination (FIG. 6, arrow).

The effect that the SSOs had on MLH3 isoforms was mirrored by the effectthe SSOs had on repeat expansion (FIG. 7). The SSO combinations andconcentrations that effectively switch MLH3 to isoform 2, also slowedGAA⋅TTC repeat expansion (compare FIGS. 6 and 7).

That splice-switching oligonucleotide directed skipping of MLH3 exon 7slows GAA⋅TTC repeat expansion in our model system provides proof ofprinciple. The SSOs were then studied in FRDA patient-derived cells. Inlight of reports of sustained morpholino action in neuronal rescuemodels in the mouse (G41, G42), whether the effect from a singleexposure to the SSOs could be sustained in post mitotic cells wasexamined.

A Single Dose of MLH3 SSOs Slows Repeat Expansion Over 4 Weeks in FRDAFibroblasts.

FRDA patient-derived fibroblastic cells do not exhibit repeat expansionat the GAA⋅TTC repeats in the FXN gene under normal circumstances.However, ectopic expression of the DNA mismatch repair protein MSH3 willcause the GAA⋅TTC repeats to expand whether the fibroblasts are passagedand actively dividing, or confluent and not dividing (G1). MSH3 wasexpressed in FRDA fibroblasts via lentivirus transduction as has beendone in the past, and then it was demonstrated that expansion by aone-time treatment with MLH3 SSOs can be reduced.

FRDA fibroblasts GM04078 (Coriell) were transduced with lentivirusexpressing MSH3 at time zero (T₀). Transduced cells were plated at highdensity and allowed to reach confluence. At week 1, some cells wereexposed to 750 nM SSOs (acceptor and donor) for 24 hours. After that,all cells were fed with normal growth media (DMEM+10% FBS). At 5 weekspost transduction (4 weeks post SSO treatment), DNA was prepared andrepeats were sized with PCR. The results of one such experiment areshown in FIG. 8. Despite the small differences, it is apparent that thetreated cells expanded less than did the untreated cells. The results ofexperiments such as this indicate that the SSOs used have a sustainedeffect on non-dividing cells, and this effect can be seen in as littleas one month in culture. Such experiments also inform our power analysisfor the planned mouse study.

Research

The “YG22” FRDA mouse model exhibits region specific GAA⋅TTC repeatexpansion (G6). MLH3-specific splice-switching oligonucleotides (SSOs)will be used to block this expansion. MLH3 has recently been identifiedas a component of CAG⋅CTG expansion in the Huntington's disease “R6”mouse model (G5) and region specific CAG⋅CTG expansion in the same modelhas been linked to MSH3 (G2-G4). The parallels are considerable; withoutbeing bound by theory, MLH3 is key to GAA⋅TTC repeat expansion in theFRDA YG22 mouse, as well as in human FRDA cells.

However, whether the SSOs will penetrate where needed and whether thevivo-morpholino versions will cause clotting will be examined in mice.Therefore, SSOs will be tested in C57BL/6J mice before moving to themuch longer experiments in the Tg(FXN)YG22 Pook mice.

The following will be tested: two types of SSO, two ages of application,and two delivery protocols in mice. Simple or “naked” morpholinos havelong been used in model organisms, including mice, and have been inhuman trials for the past several years (G7-G9). Their safety andefficacy are well known. One drawback is the consistent penetration inadult tissue.

The conjugated “vivo-morpholino” is designed to have better tissuepenetration in the mouse (G40, G43). Without being bound by theory,retrograde transport of vivo-morpholinos will get them into dorsal rootganglia. The vivo-morpholinos are superior for tissue culture comparedto naked morpholinos (G41, G44).

Design and Test of MLH3 Exon Skipping SSOs in Mouse Cells.

The mouse MLH3 exon structure parallels that of humans. The SSOs andsplice assay nucleotides needed will parallel those used for the humanMLH3 locus, although they will have somewhat different nucleotidesequences. These reagents will be tested in mouse cell lines in culture,including cells from the C57BL/6 mouse, just as has been done with humanMLH3. A small synthesis of vivo-morpholinos will be used for the tissueculture assays. Once mouse MLH3 isoform switching has been optimized, alarger synthesis of the vivo-morpholinos and the corresponding simplemorpholinos will be ordered. When SSOs for the mouse are discussed inthis example, either naked or vivo-morpholinos, it will most likelyrefer to a cocktail of donor and acceptor blocking sequences as shown inFIGS. 6 and 7.

SSOs that are effective for mouse MLH3 will be identified quickly.Confidence in the bioinformatic analysis of the MLH3 gene in mice, thedesign of the SSOs and the splice detection oligonucleotide pairs hasbeen established.

Breeding of Mice During the Design and Testing Period.

B6.Cg-Fxn^(tm1MKn)-Tg(FXN)YG22Pook/J and C57BL/6J mice will be orderedfrom the Jackson Laboratory. The strainB6.Cg-Fxn^(tm1Mkn)Tg(FXN)YG22Pook/J is a double mutant: hemizygous for ahuman FXN locus with expanded GAA⋅TTC repeats and heterozygous for aknockout of the mouse FXN locus. Through selective breeding, micehomozygous for the human FXN locus, but lacking the knockout of mouseFXN, will be produced. Subsequent crosses with C57BL/6J mice willproduce offspring bearing a human FXN transgene and normal mouse FXNalleles to avoid possible selection against repeat expansion due toinsufficient frataxin.

Initial Testing of SSO Safety and Efficacy in C57BL/6 Mice.

It can be quickly ascertained in wild type mice whether the SSOs aresafe and effective for MLH3 isotype switching. After a single injectionof candidate SSOs in newborn (PND0, in facial vein) or adult (8 weeks,in tail vein) C57BL/6J mice, the mice will be humanely killed anddissected after intervals (see Table 3 for numbers, dosing andintervals). RNA will be isolated from brain, cerebellum, dorsal rootganglia, heart, and liver, and will be assayed for MLH3 isotype viareverse transcription PCR (RT-PCR). Safety, tissue penetration, andpersistence of action by high or low doses of naked morpholino andvivo-morpholino SSOs will be determined in both newborn and adult micebefore any experiments in the “YG22” mice are conducted.

TABLE 3 Mouse distribution and end points in research protocol. DNA andRNA will be isolated from brain, cerebellum, dorsal root ganglia, heartand liver. Mice Regimen Reagent Dose Collection time points 66 wt micePost natal day 0 12 vivo- 50 mg/kg RNA will be isolated at weeksC57BL/6J (PND0) Cohort morpholino 1, 2, 4 and 8 for splice One-timeinjection 12 morpholino 50 mg/kg switching efficacy RT-PCR 12 controlSaline assay (N = 3 for each time point & condition). Young adult Cohort12 vivo- 50 mg/kg RNA will be isolated 1 and 2 Single injection at 8morpholino 5 mg/kg weeks after injection for splice weeks 12 morpholino50 mg/kg switching efficacy RT-PCR 5 mg/kg assay (N = 3 for each time &6 control Saline condition). 96 “YG22” Post natal day 0 16 vivo- 50mg/kg DNA & RNA will be isolated Transgenic (PND0) Cohort morpholino at3 months (n = 8) and mice One-time injection 16 morpholino 50 mg/kg at 6months (n = 8) carrying 16 control Saline Tg(FXN)YG Young adult Cohort16 vivo- 50 mg/kg 8 DNA & RNA @6 months 22Pook Bimonthly injectionmorpholino 5 mg/kg 8 DNA & RNA @6 months starting at 8 weeks 16morpholino 50 mg/kg 8 DNA & RNA @6 months 5 mg/kg 8 DNA & RNA @6 months16 control Saline 16 DNA & RNA @6 months

Two Types of SSO and Two Delivery Protocols to be Tested in YG22 Mice.

A single application at birth (post natal day 0, PND0) will beadministered via facial vein injection. Application of naked morpholinoor vivo-morpholino SSOs at birth has been shown to penetrate into thecentral nervous system (CNS), possibly due to a leaky blood brainbarrier at birth (G41, G42). For the PND0 cohort, a one-time injectionwill be followed by an interval of normal mouse rising with no furtherexperimental manipulations until the mouse is humanely killed at theappropriate time point (see Table 3).

Bi-monthly application in adult mice, starting at 8 weeks will beadministered via tail vein injection. The “YG22” cohort of GAA⋅TTCrepeat bearing adult mice will get a tail vein injection every two weeksstarting at 8 weeks before the mice are humanely killed at 26 weeks ofage. Further, in adult mice we will test two concentrations of SSO, 5mg/kg and 50 mg/kg (see Table 3 for distribution). A chronic low dosemay be safer than a high dose, and these experiments will help determineif the low dose is sufficient to get into tissues such as the heart(G45, G46).

SSO efficacy will be assayed in two ways. The MLH3 isoform 1 to isoform2 ratios will be measured via RT-PCR. GAA⋅TTC repeat length in “YG22”mice will be measured via PCR. Tissue from the CNS and PNS, as well assomatic tissues, will be further assayed.

Tissues to be dissected from mice for RNA and DNA isolation willinclude 1) brain, 2) cerebellum, 3) dorsal root ganglia (DRG), 4) heartand 5) liver. Tail DNA will also have been prepared separately forgenotyping, and may serve as an additional control.

Without being bound by theory, reduced expansion in the cerebellum andDRG of PND0 injected YG22 mice will be observed. Without being bound bytheory, persistent SSO activity will be observed in tissues containinglong lived post-mitotic cells such as brain, cerebellum and heart, butless so in liver due to dilution by cell division.

Without being bound by theory, robust SSO activity will be observed inthe livers of the young adult YG22 repeated high dose morpholino andvivo-morpholino cohorts, and a little less activity will be observed inthe heart.

Without being bound by theory, little or no SSO activity will beobserved in the heart and little activity will be observed in the liversof low dose morpholino YG22 adult cohort, but moderate to robustactivity will be observed in the low dose vivo-morpholino YG22 adultcohort due to chronic accumulation of the vivo-morpholino.

Without being bound by theory, SSO activity will be observed in the DRGof vivo-morpholino, but not naked morpholino treated adults. Ifsufficient GAA⋅TTC expansion occurs in the DRG, an effect of SSOtreatment, will be observed, particularly in the high dosevivo-morpholino cohort.

Without being bound by theory, reduced expansion will not be observed inthe cerebellum or brain of the young adult YG22 cohort due to poorpenetration of the blood brain barrier no matter the dose.

Statistics

Statistical analysis will predominantly use one-way or two-way ANOVA. Weused power analysis to arrive at the number of animals for the study,based upon our ability to separate repeats differing by 1-2% of length.The transgenic repeat lengths detected by PCR will range between 1000 to1300 base pairs (bp). FRDA patient derived cells we have worked with cangain 4 to 6 repeats (12-18 bp) a month. We assume that if the mousecerebellar samples gain comparably, we should detect a 50% effect of thetreatment in 2 months with 8 (morpholino) 8 (vivo-morpholino) to 16controls for each injection/dosing condition with 80% power. If mouseGAA⋅TTC repeat expansion exceeds the human rate, we will have morepower. Our long-term experiments should have a larger expansiondifferential producing more power and allowing for some dropout. TheRT-PCR based mRNA splicing assay is more easily measured, has a largedelta and requires a smaller “n” when not accompanied by the DNA repeatsizing assay. Hence the wild-type C57BL/6J mice used in early tests havereduced numbers compared to “YG22.”

Example 3

Monitoring mice for long-term consequences of the treatments describedin these examples can be done. The longest experiments will look at6-month-old mice, well before even a complete knockout from birth wouldshow an effect. Changing the ratio of MLH3 isoforms, not knocking MLH3out, will be changed. Furthermore, morpholino oligomers are lost ordiluted by cell division. Consequently, cells at risk for a cancerphenotype such as intestinal epithelia or lymphoblasts will onlytransiently be deficient in MLH3 isoform 1.

In one embodiment, the frataxin deficient phenotype of the Pook mice canbe studied at a later stage. The mFXN knockout allele will be bred outto avoid potential interference with repeat expansion by a frataxindeficient phenotype. Here, frataxin replete Tg(FXN)YG22Pook mice will beused solely to examine repeat expansion.

As discussed in the preceding examples, SSO efficacy will be assayed intwo ways: 1) the MLH3 isoform 1 to isoform 2 ratios will be measured viaRT-PCR. This will be done in cultured cells, C57BL/6J and YG22Pook mice;and 2) GAA⋅TTC repeat length in YG22Pook mice will be measured via PCR(conventional and small-pool).

It will be determined whether morpholino SSO mediated MLH3 isoformswitching can reduce the rate of GAA⋅TTC repeat expansion in theYG22Pook mice. Because morpholino SSOs only need to bind their target toabrogate splicing, an SSO or cocktail of SSOs effective in mice will beidentified. Without being bound by theory, the most effective SSO or SSOcocktail found in tissue culture can be toxic to mice in thevivo-morpholino form, but the unmodified, or simple morpholinooligomers, will also be used as a backup. The simple morpholino SSOswill have reduced penetration in adult tissue. But, simple morpholinoSSOs have been shown to be effective from a single PND0 injection inmice for at least 65 days (2) and are safe enough that several are inhuman trials (11-13).

Testing of the SSOs can be done quickly and relatively inexpensive incultured mouse cells and wild-type C57BL/6J mice, while the YG22Pookmice are bred. Without being bound by theory, both immediate toxicityand the efficacy in MLH3 splice switching in C57BL/6J tissues will bedetermined in a matter of days. The longer time points are there todetermine the staying power of the morpholinos in various tissues todetermine the dosing regimen needs to be adjusted before startinglong-term experiments in YG22Pook mice.

MLH3 isoform 1→2 switching is a direct measure of SSO efficacy.Consequent effects of a decrease in MLH3 isoform 1 on GAA⋅TTC repeatexpansion rate will be measured via PCR across the repeat (bothconventional and small pool PCR).

The mice available from Jackson lab are heterozygous for mFXN knockoutand hemizygous for Tg(FXN)YG22Pook. Breeders will be developed withnormal mFXN genes that are homozygous for Tg(FXN)YG22Pook for tworeasons: (1) Breeding with C57BL/6J will produce offspring that are allhemizygous for YG22 so that mice can be efficiently treated at birthwithout wasting reagent or waiting for genotyping; and (2) Normal mousefrataxin expression will ensure that expansion of the repeat will not becounter-selected by frataxin insufficiency.

RT-PCR determination of MLH3 isoform 1→2 switching is a direct measureof SSO efficacy, and comparison of MLH3 isoforms to those in controltissues will be informative regarding tissue penetration of the SSOs.

Mice available through the Jackson Laboratory, which are heterozygousfor mFXN knockout and hemizygous for Tg(FXN)YG22Pook, will be used.Breeders will be developed with normal mFXN genes that are homozygousfor Tg(FXN)YG22Pook for two reasons: 1) Breeding with C57BL/6J willproduce offspring that are all hemizygous for YG22 so that mice can beefficiently treated at birth without wasting reagent or waiting forgenotyping; and 2) Normal mouse frataxin expression will ensure thatexpansion of the repeat is not counter-selected by frataxininsufficiency.

Without being bound by theory, the activity of the SSOs will diminishover a shorter time course in rapidly dividing tissues as the mousegrows and the SSOs will be diluted. Furthermore, cells that arepost-mitotic at birth should retain SSOs and their activity far longer.For instance, Porensky et al. showed that a single injection of simplemorpholino SSOs at birth lead to splice switching that remained robustin brain and spinal cord at 65 days (2). Consequently, there will alsobe sustained activity of PND0 administered SSOs in brain and spinal cordfor at least 8 weeks. If the splice switching activity declinesthereafter, the aggregate effects of diminished and partially diminishedMLH3 isoform 1 expression on GAA⋅TTC repeat expansion at 3 months orgreater will be able to be assessed.

The heart will be examined because it is an affected tissue in FRDA.Without being bound by theory, the working model will show that therepeat focally expands in heart leading to stochastic loss of fibers(along with their expanded repeats). Thus, it is nevertheless of greatinterest to determine whether therapeutic SSOs can be administered intothe heart.

Changes in the rate of expansion will be assessed by comparing the rateof expansion in tissues that typically exhibit expansion with those thattypically do not in YG22 mice. In addition, the rate of expansion willbe compared in tissues from untreated littermates to tissues in thetreatment groups. Conventional PCR has been shown to be sufficient tosee gross changes in size in the YG22 repeat, particularly in thecerebellum (A14, A15). Small pool PCR will have to be used to assessmore rare events in other tissues, as well as to simplify the smearobtained from expanded repeats seen with conventional PCR in thecerebellum (A16).

REFERENCES

-   A1. Summerton, J. and Weller, D. (1997) Morpholino antisense    oligomers: design, preparation, and properties. Antisense Nucleic    Acid Drug Dev, 7, 187-195.-   A2. Porensky, P. N., Mitrpant, C., McGovern, V. L., Bevan, A. K.,    Foust, K. D., Kaspar, B. K., Wilton, S. D. and Burghes, A. H. (2012)    A single administration of morpholino antisense oligomer rescues    spinal muscular atrophy in mouse. Human molecular genetics, 21,    1625-1638.-   A3. Morcos, P. A., Li, Y. and Jiang, S. (2008) Vivo-Morpholinos: a    non-peptide transporter delivers Morpholinos into a wide array of    mouse tissues. BioTechniques, 45, 613-614, 616, 618 passim.-   A4. Ferguson, D. P., Dangott, L. J. and Lightfoot, J. T. (2014)    Lessons learned from vivo-morpholinos: How to avoid vivo-morpholino    toxicity. BioTechniques, 56, 251-256.-   A5. Halabi, A., Ditch, S., Wang, J. and Grabczyk, E. (2012) DNA    mismatch repair complex-   MutSbeta promotes GAA⋅TTC repeat expansion in human cells. The    Journal of biological chemistry, 287, 29958-29967.-   A6. Hienonen, T., Laiho, P., Salovaara, R., Mecklin, J. P.,    Jarvinen, H., Sistonen, P., Peltomaki, P., Lehtonen, R.,    Nupponen, N. N., Launonen, V. et al. (2003) Little evidence for    involvement of MLH3 in colorectal cancer predisposition. Int J    Cancer, 106, 292-296.-   A7. Liu, H. X., Zhou, X. L., Liu, T., Werelius, B., Lindmark, G.,    Dahl, N. and Lindblom, A. (2003) The role of hMLH3 in familial    colorectal cancer. Cancer Res, 63, 1894-1899.-   A8. Lipkin, S. M., Moens, P. B., Wang, V., Lenzi, M., Shanmugarajah,    D., Gilgeous, A., Thomas, J., Cheng, J., Touchman, J. W.,    Green, E. D. et al. (2002) Meiotic arrest and aneuploidy in    MLH3-deficient mice. Nature genetics, 31, 385-390.-   A9. Chen, P. C., Dudley, S., Hagen, W., Dizon, D., Paxton, L.,    Reichow, D., Yoon, S. R., Yang, K., Arnheim, N., Liskay, R. M. et    al. (2005) Contributions by MutL homologues Mlh3 and Pms2 to DNA    mismatch repair and tumor suppression in the mouse. Cancer Res, 65,    8662-8670.-   A10. Chen, P. C., Kuraguchi, M., Velasquez, J., Wang, Y., Yang, K.,    Edwards, R., Gillen, D., Edelmann, W., Kucherlapati, R. and    Lipkin, S. M. (2008) Novel roles for MLH3 deficiency and TLE6-like    amplification in DNA mismatch repair-deficient gastrointestinal    tumorigenesis and progression. PLoS genetics, 4, e1000092.-   A11. Kinali, M., Arechavala-Gomeza, V., Feng, L., Cirak, S., Hunt,    D., Adkin, C., Guglieri, M., Ashton, E., Abbs, S.,    Nihoyannopoulos, P. et al. (2009) Local restoration of dystrophin    expression with the morpholino oligomer AVI-4658 in Duchenne    muscular dystrophy: a single-blind, placebo-controlled,    dose-escalation, proof-of-concept study. Lancet Neurol, 8, 918-928.-   A12. Goemans, N. M., Tulinius, M., van den Akker, J. T., Burm, B.    E., Ekhart, P. F., Heuvelmans, N., Holling, T., Janson, A. A.,    Platenburg, G. J., Sipkens, J. A. et al. (2011) Systemic    administration of PRO051 in Duchenne's muscular dystrophy. The New    England journal of medicine, 364, 1513-1522.-   A13. Cirak, S., Arechavala-Gomeza, V., Guglieri, M., Feng, L.,    Torelli, S., Anthony, K., Abbs, S., Garralda, M. E., Bourke, J.,    Wells, D. J. et al. (2011) Exon skipping and dystrophin restoration    in patients with Duchenne muscular dystrophy after systemic    phosphorodiamidate morpholino oligomer treatment: an open-label,    phase 2, dose-escalation study. Lancet, 378, 595-605.-   A14. Anjomani Virmouni, S., Sandi, C., Al-Mandawi, S. and    Pook, M. A. (2014) Cellular, Molecular and Functional    Characterisation of YAC Transgenic Mouse Models of Friedreich    Ataxia. PloS one, 9, e107416.-   A15. Ezzatizadeh, V., Sandi, C., Sandi, M., Anjomani-Virmouni, S.,    Al-Mandawi, S. and Pook, M. A. (2014) MutLalpha heterodimers modify    the molecular phenotype of Friedreich ataxia. PloS one, 9, e100523.-   A16. Clark, R. M., De Biase, I., Malykhina, A. P., Al-Mandawi, S.,    Pook, M. and Bidichandani, S. I. (2007) The GAA triplet-repeat is    unstable in the context of the human FXN locus and displays    age-dependent expansions in cerebellum and DRG in a transgenic mouse    model. Human genetics, 120, 633-640.

Example 4

Exploring DNA Mismatch Repair Complexes Involved in Repeat Expansion.

Friedreich ataxia (FRDA) is a progressive neurodegenerative disordercaused by GAA. TTC repeat expansion in the first intron of the frataxin(FXN) gene. Disease severity correlates to the length of the expandedrepeats and the reduction of FXN mRNA. The mechanism of repeat expansionis not yet completely understood; however, it has previously been shownthat the expansion rate is associated to transcription within therepeats. Without being bound by theory, DNA repair enzymes are attractedto structures formed within the GAA. TTC repeat during transcription andthe subsequent actions of these repair enzymes then promote theexpansion process. It has been shown that shRNA knockdown of either MSH2or MSH3, components of mismatch repair complex MutSβ, slowed GAA⋅TTCexpansion in the model and FRDA patient fibroblasts. Furthermore,ectopic expression of MutSβ induced GAA. TTC repeat expansion in thenative FXN gene. Other components of DNA mismatch repair complexes arebeing examined to elucidate their role in FRDA (for example, see FIGS.2A-B and FIG. 3 for MLH1, MLH3, and PMS2 data). For example, componentsof the heterodimeric complexes in mismatch repair's human homolog MutLare of interest, which participate in the repair of a subset ofmismatches, recognized by the MSH2-MSH3 complex. Due to alternativesplicing and alternative AUG start codon usage there are many possiblevariations in MutL complexes (See FIG. 9B). Without being bound bytheory, only a few particular isoforms of these complexes areresponsible for repeat expansion. Presently, these components of DNAmismatch repair are being examined in order to elucidate their role inFRDA and discover possible therapeutic targets, exploring the role ofMLH3 isoforms in FRDA repeat expansion. The heterodimer of MutLHomologue one (MLH1) with MLH3 forms the necessary MutL complex, whichis known as MutLγ. MLH3, a component of the MutLγ complex, has 2isoforms due to alternative splicing. MLH3 isoform 1 (MLH3 iso1) hasexon 7 and MLH3 iso2 lacks exon 7 (FIG. 9B). Exon 7 contains theendonuclease domain. Without being bound by theory, excluding exon 7 canapproximate a functional knock out if MLH3 is critical to repeatexpansion. The expression levels of the spliced variants were correlatedto the GAA⋅TTC repeat expansion rate in a human cell model.

Detecting MLH3 Isoforms 1 and 2.

To study the functional diversity of MLH3 protein isoforms, the alteredexpression levels of the spliced variants were measured to evaluatetheir effects on GAA⋅TTC repeat expansion. Primer pairs, MLH3 L3324 andMLH3 R3757, resulted in a 434 bp band for MLH3 iso1 and a 362 bp bandfor MLH3 iso2. A 16.6% difference allowing for visualizing the presenceor absence of the 72 nucleotide long exon 7. Dilutions of definedisoform templates containing or excluding exon 7 were done in 10:1, 1:1,and 1:10 respectively to demonstrate the quantitative nature of this PCR(FIG. 9A).

Forcing Exon Choice.

Splice switching oligos (SSOs) were designed to mask the acceptor anddonor regions of MLH3 exon 7 to induce skipping of exon 7 and theconsequent production of MLH iso2 (FIG. 5).

RT-PCR Demonstrates Exon Skipping.

100 nM and 500 nM dilutions of acceptor, donor, and a combination ofboth SSOs were given twice a week to FRDA model cells to examine theeffect of MLH3 SSOs on MLH3 isoforms. The control received media only.Cells were assessed for MLH3 isoform variant expression with RT-PCRusing the designed primer pair (FIG. 6). The control resembles the 10:1(MLH1 iso1:MLH3 iso2) ratio of defined templates of known concentrationshown in FIG. 9A. The combination of acceptor and donor SSOs caused theeffective exclusion of exon 7.

MLH3 Iso1 Required for Expansion.

PCR analysis of GAA⋅TTC expansion was conducted at week 3 with 100 nMand 500 nM dilutions of acceptor, donor, and a combination of both SSOsto examine the effect of MLH3 SSOs on MLH3 isoforms. Repeat growth assayPCR product equals 500 bp flanking sequence plus 3×'s number of repeats.Sample 6 with 500 nM of both acceptor and donor SSOs slowed expansion(FIG. 7). Sample 4 with 500 nM of the donor SSO also had a slowingeffect but not as substantially as the combination of the SSOs (FIG. 7).

MLH3 contributes to GAA⋅TTC repeat expansion in human cells.Specifically, MLH3 exon 7 is necessary for GAA⋅TTC repeat expansion inhuman cells. Lack of exon 7 slows GAA⋅TTC repeat expansion in FRDA modelcells. The endonuclease domain of MLH3 is needed for this effect.

Targeting both the splice donor and acceptor region of exon 7 excludedMLH3 iso1. Such a method as splice skipping can have potential as afuture therapeutic avenue for FRDA. In one embodiment, MLH3 is useful asa therapeutic target to slow the progression of repeat expansiondisorders such as Friedreich ataxia. On another embodiment, smallmolecule directed skipping of MLH3 exon 7 is a useful therapeuticapproach.

Future studies will include: (1) Observation of intrinsic expression ofMLH3 variants iso1 and iso2 in different cell lines, including FRDApatient cells; (2) Observation of variants in the CNS and heart tissue;and (3) Explore MLH1, which has 22 isoforms, and is the MutLγ partner ofMLH3.

Example 5

The core innovation of this technology is a therapeutic oligonucleotidefor the treatment of DNA repeat expansion diseases, which includeFriedreich ataxia, ALS, and Huntington's disease, among others. Thisinnovation will likely be given orphan drug designation, highlighting acommercialization strategy associated with a number of commercialbenefits.

Friedreich ataxia (FRDA) is a progressive neurodegenerative disordercaused by GAA. TTC repeat expansion in the first intron of the frataxin(FXN) gene. Disease severity correlates to the length of the expandedrepeats and the consequent reduction of FXN gene expression. While themechanism of repeat expansion is not fully understood, it has been shownthat the expansion rate is associated with transcription within therepeat (FIGS. 1A-F) and requires the action of MutSbeta and a MutLcomplex (G1, G2). The necessary complex is MutLgamma, a heterodimer ofMLH1 and MLH3 (see FIG. 1E). MLH3 expression is key to DNA repeatexpansion in human cells. MLH3 is expressed in humans as two isoforms.MLH3 isoform 1 includes exon 7, while MLH3 isoform 2 lacks exon 7 (FIG.5). Skipping exon 7 leaves MLH3 isoform 2 intact, and does not impactthe cellular ratios of MLH1 and its binding partners PMS2, PMS1 andMLH3. Splice-Switching Oligonucleotides (SSOs) described herein directedskipping of MLH3 exon 7 and slowed GAA⋅TTC repeat expansion in thismodel system, and is proof of principle as a therapy (see FIGS. 6-7).

Currently there is no effective treatment and no cure for any of themany DNA repeat expansion diseases. The core innovation of thistechnology aims to markedly shift the therapeutic focus from purelysymptomatic to one that directly tackles the underlying diseasemechanism. By slowing the expansion rate of the disease-causing DNArepeat, this therapeutic aims to slow the progression of the disease andextend a high quality of life for the individual.

Without being bound by theory, the approach targets a central mechanismthat is shared by a number of repeat expansion diseases. Therefore, ithas the potential to treat many, if not all of the diseases in thisclass. The gene-specific exon skipping in Duchenne muscular dystrophy(DMD) is more limited; SSO mediated exon skipping in DMD can only treata fraction of the DMD patient population (G3-G5). Nonetheless, at leasttwo startup companies (Sarepta Therapeutics and Prosensa) have beenformed around exon skipping in DMD. Diseases that can benefit from thistechnology include Friedreich ataxia, ALS (c9orf72), Huntington'sdisease, Fragile X syndrome, Myotonic dystrophy Types I and II, SpinoCerebellar Ataxias (SCAs) currently including SCA1, SCA2, SCA3, SCA6,SCAT, SCAB, SCA10, SCA12, SCA17, SCA31 and SCA36 among others.

Explanation of Specific Experiments and Concepts to be Proven

The mismatch repair protein MLH3 is key to GAA⋅TTC repeat expansion.This minor component of MMR will be developed as a therapeutic target tolimit repeat expansion in FRDA patients, and all repeat expansionpatients. The SSOs used are the same type already in human trials forDuchenne muscular dystrophy (G3-G5). The work shown in FIG. 6 and FIG. 7were carried out using the “tandem reporter” model system (G10).

The work will be expanded upon, verifying it in primary patient cells,adding additional disease causing repeats to the “tandem reporter”expansion model such as CAG⋅CTG (DM and the polyglutamine disorders),CGG⋅CCG (fragile X) and CCGGGG⋅CCCCGG (ALS due to C9ORF72), anddesigning and testing additional SSOs to achieve greater efficacy. Theapproach will also be studied using a mouse model of Friedreich ataxiarepeat expansion, as discussed in the Examples herein.

MLH3 Splice Skipping Stops Repeat Expansion in Patient-Derived Cells

FRDA patient-derived cells do not exhibit repeat expansion at theGAA⋅TTC repeats in the FXN gene under normal circumstances. However,ectopic expression of the DNA mismatch repair protein MSH3 can cause theGAA⋅TTC repeats to expand (G2). MSH3 works upstream of MLH3 in a minorarm of mismatch repair (see FIGS. 1A-F). MSH3 will be expressed intarget cells as has been done in the past, and then it will bedemonstrated that the repeat can be stopped from expanding by treatingthe cells with MLH3 specific SSOs. Friedreich ataxia patient cells gainan average of 1 repeat a week, so after 6 to 8 weeks of continuousculture. Without being bound by theory, a positive result in the repeatsize profiles of the collected DNA, as in the “tandem reporter” model(FIG. 7), will be observed.

Myotonic dystrophy (DM1) patient-derived cells will be obtained from theCoriell cell repositories, and ectopic expression of MSH3 will be usedto encourage the repeats to expand as outlined in Halibi et al (G2). Thelong CTG⋅CAG repeats in the DMPK gene of DM1 are fairly unstable inpatients. Expansion will be detected, although the exact time course isnot yet known. A recent publication on a mouse model of DM1 suggeststhat the rate of CTG⋅CAG expansion in mouse cells (G11) exceeds what wefound for GAA⋅TTC in FRDA patient-derived cells. However, the bona fideDM1 patient-derived cells may expand more slowly, so the cells will becultured for 60 to 120 days before collecting DNA.

Fragile X syndrome patient and carrier-derived cells will be obtainedfrom the Coriell cell repositories, and ectopic expression of MSH3 willbe used to encourage the CGG⋅GGC repeats to expand as outlined in Halibiet al (G2). Several mouse models of Fragile X syndrome have beendeveloped (G12,G13) and PCR techniques incorporating 2M to 3M betaineare known (G12) that will get through these templates to size therepeats accurately after 60 to 120 days of continuous culture.

Repeat Tracts Representative of Repeat Expansion Diseases of Interestinto Tandem Reporter Cell Lines Will be Cloned to Test MLH3 ExonSkipping

Most of the repeat expansion diseases do not have suitable patientderived cell lines with which to perform expansion studies. The rapidexpansion system with a repeat cloned between tandem reporters expandsGAA⋅TTC repeats at a rate ten- to twenty-fold faster than the patientderived cells that have been enhanced with MSH3 expression (G2). Thecell lines are permissive for GAA⋅TTC expansion. Therefore, tandemreporter constructs will be made as have been done previously forGAA⋅TTC tracts (G1) using the serial ligation method previouslydeveloped (G14). Huntington's CAG⋅CTG lines, DM1 CTG⋅CAG lines, FragileX CGG⋅CCG lines and ALS CCGGGG⋅CCCCGG lines will be generated. Thetechniques for making the cell lines are routine in the inventor'slaboratory (e.g., G1 and G14 describe the processes in detail, which areherein incorporated by reference in their entireties).

Generation of these additional disease model cell lines will demonstratethe generality of the expansion model to other repeat expansiondiseases. Comparing the rates of expansion of these various repeats inan identical environment will go far in elucidating the underlyingmechanisms. The rapid expansion system with the tandem reporter carryingvarious disease specific DNA repeats may be desired as discoveryplatforms by companies seeking to develop additional therapies forrepeat expansion.

Design and Test of Additional SSOs to Optimize MLH3 Exon Skipping

The results shown in FIG. 6 indicate that while the initial choices foracceptor and donor SSOs are very effective in combination, there is roomfor improvement. This is particularly true for the acceptor blockingSSO. New SSOs will be designed, particularly at the acceptor site, toimprove efficacy. Safety and efficacy studies will also be performed ina mouse model of Friedreich ataxia GAA⋅TTC repeat expansion. Severalsets of mouse-specific SSOs will be tested to optimize mouse MLH3 exonskipping. The data obtained from the mouse studies will inform thesearch for a better human set of SSOs.

Goals

Demonstration of the efficacy of MLH3 exon skipping in bona fide FRDApatient cells will be conducted. Cell lines will be obtained, grown for60 days plus or minus ectopic MSH3 and SSOs, and subsequently willperforming the RT-PCR assays on the MLH3 splice isoforms and the PCRreaction to determine repeat lengths.

Goals will be: Preparing the plasmids for the in vitro constructions.Selecting for the site-specific integration of the constructs,amplifying the cells in tissue culture and then assaying the initialsize of the repeats before freezing the cell lines in liquid nitrogen.If the patient derived cells for myotonic dystrophy or Fragile Xsyndrome do not produce results, the corresponding repeat will be usedin the rapid expansion system with the MLH3 SSOs to provide the data.

REFERENCES

-   G1. Ditch, S., Sammarco, M. C., Banerjee, A. and Grabczyk, E. (2009)    Progressive GAA⋅TTC repeat expansion in human cell lines. PLoS    genetics, 5, e1000704.-   G2. Halabi, A., Ditch, S., Wang, J. and Grabczyk, E. (2012) DNA    mismatch repair complex MutSbeta promotes GAA⋅TTC repeat expansion    in human cells. The Journal of biological chemistry, 287,    29958-29967.-   G3. Kinali, M., Arechavala-Gomeza, V., Feng, L., Cirak, S., Hunt,    D., Adkin, C., Guglieri, M., Ashton, E., Abbs, S.,    Nihoyannopoulos, P. et al. (2009) Local restoration of dystrophin    expression with the morpholino oligomer AVI-4658 in Duchenne    muscular dystrophy: a single-blind, placebo-controlled,    dose-escalation, proof-of-concept study. Lancet Neurol, 8, 918-928.-   G4. Goemans, N. M., Tulinius, M., van den Akker, J. T., Burm, B. E.,    Ekhart, P. F., Heuvelmans, N., Holling, T., Janson, A. A.,    Platenburg, G. J., Sipkens, J. A. et al. (2011) Systemic    administration of PRO051 in Duchenne's muscular dystrophy. The New    England journal of medicine, 364, 1513-1522.-   G5. Cirak, S., Arechavala-Gomeza, V., Guglieri, M., Feng, L.,    Torelli, S., Anthony, K., Abbs, S., Garralda, M. E., Bourke, J.,    Wells, D. J. et al. (2011) Exon skipping and dystrophin restoration    in patients with Duchenne muscular dystrophy after systemic    phosphorodiamidate morpholino oligomer treatment: an open-label,    phase 2, dose-escalation study. Lancet, 378, 595-605.-   G6. The New Economics of Orphan Diseases. Genetic Engineering &    Biotechnology News, Jan. 1, 2013.-   G7. The Economic Power of Orphan Drugs. Thomson Reuters, 2012.-   G8. Top 20 orphan drugs by 2018. FiercePharma, Jul. 23, 2013.-   G9. Global Market for Orphan Drugs is Expected to Reach $112 Billion    in 2017. Drugs.com, August 2013.-   G10. Banerjee, A., Sammarco, M. C., Ditch, S., Wang, J. and    Grabczyk, E. (2009) A novel tandem reporter quantifies RNA    polymerase II termination in mammalian cells. PloS one, 4, e6193.-   G11. Gomes-Pereira, M., Hilley, J. D., Morales, F., Adam, B.,    James, H. E. and Monckton, D. G. (2014) Disease-associated CAG.CTG    triplet repeats expand rapidly in non-dividing mouse cells, but cell    cycle arrest is insufficient to drive expansion. Nucleic acids    research.-   G12. Lavedan, C., Grabczyk, E., Usdin, K. and Nussbaum, R. L. (1998)    Long uninterrupted CGG repeats within the first exon of the human    FMR1 gene are not intrinsically unstable in transgenic mice.    Genomics, 50, 229-240.-   G13. Entezam, A., Biacsi, R., Orrison, B., Saha, T., Hoffman, G. E.,    Grabczyk, E., Nussbaum, R. L. and Usdin, K. (2007) Regional FMRP    deficits and large repeat expansions into the full mutation range in    a new Fragile X premutation mouse model. Gene, 395, 125-134.-   G14. Grabczyk, E. and Usdin, K. (1999) Generation of microgram    quantities of trinucleotide repeat tracts of defined length,    interspersion pattern, and orientation. Analytical biochemistry,    267, 241-243.-   G15. BioMarin buys Prosensa for up to $840M, shoots for quick OK of    Duchenne drug. FierceBiotech, Nov. 24, 2014.

Example 6

Summary

Without being bound by theory, the GAA⋅TTC repeats that cause Friedreichataxia (FRDA) continue to grow in length over time in the tissues thatare affected by the disease. Without being bound by theory, this is whatcauses the gradual onset of Friedreich ataxia, and also what causes itsprogressive nature. Data indicate that this continued expansion ofGAA⋅TTC repeats requires transcription through the repeat then thesequential actions of several DNA mismatch repair proteins calledMutSbeta (MSH2/MSH3 heterodimer) and then MutLgamma (MLH1/MLH3heterodimer). Transcription is the process of copying the doublestranded DNA into RNA so that protein can be made. Subjects, such ashumans, need transcription of the frataxin gene. However, duringtranscription of the GAA⋅TTC repeat, the repetitive DNA can becomemisaligned. A small loop in the misaligned DNA can be mistaken for amismatch by MutSbeta, which binds it, and then attracts MutLgamma.MutLgamma is the protein complex that cuts the DNA in the repeat tostart the expansion. Without the cut, there is no expansion. One smallpart of MLH3, called exon 7, is the knife that does the cutting. Inpeople there are two forms of MLH3, one carries the knife (exon 7), onedoes not. As described herein, compositions and methods designed to skipexon 7 have been identified, using splice-switching oligonucleotides(SSOs), so that little or no MLH3 carries a knife. The repeat stopsexpanding in cells that are treated with SSOs. The mouse MLH3 gene(mMlh3) is like that of humans (hMLH3). There is a mouse model of FRDAcalled “YG-22” that shows tissue specific GAA⋅TTC repeat expansion. Asdescribed herein, testing SSOs to block this expansion in mice serves asa first step heading to human trials.

First, the mouse MLH3 gene was targeted with a panel of SSOs specific tothe mouse. This experiment was completed, and the SSOs were tested forefficiency of splice switching in mouse cell lines. The mouse cell linesthat were initially used turned out to express little mMlh3, which madethe experiments difficult and time consuming. Subsequently, a number ofmouse cell lines were tested to find ones that were more like neurons.One of these neuron-like cell lines expressed sufficient mMlh3 todetermine that an SSO pair flanking the knife exon of mMlh3 would workmuch like the human SSOs for hMLH3. Subsequent experiments tested theseSSOs in the mice. The first hurdle in the mice was a safety concern. Inrare cases that are sequence-specific, SSOs can clump together and causea blood clot in mice. Initial tests in mice demonstrate that this hasnot happened, and that the morpholinos were all well tolerated.Subsequent, tests will show how well these SSOs are at splice switchingmMlh3 in different mouse tissues and organs, and also the ability of theSSOs to slow repeat expansion in FRDA model mice.

MLH3 is expressed in humans as two isoforms, MLH3 isoform 1 and MLH3isoform 2, due to alternative splicing. MLH3 isoform 1 includes exon 7,which contains a conserved endonuclease domain, while MLH3 isoform 2lacks exon 7. It was recently determined that the MLH3 isoform 1 isrequired for GAA⋅TTC expansion, while isoform 2 is not. Skipping exon 7by use of SSOs effectively shifts MLH3 to isoform 2 and stops repeatexpansion in human cells. Finally, skipping exon 7 leaves MLH3 isoform 2intact, so the SSOs will not impact the total cellular ratios of MLH1and its binding partners PMS2, PMS1 and MLH3.

Forced Exclusion of the Exon Coding for the Mouse MLH3 EndonucleaseDomain, as Well as Neighboring Exons as a Backup.

The mouse MLH3 exon structure parallels that of humans except that theendonuclease domain is contained in exon six rather than exon seven. InFIG. 11 this exon is circled in red to highlight it. Although mouse MLH3is not reported to have isoforms lacking this exon, the exon is 72 baseslong, and skipping it leaves the downstream exons in the sametranslational reading frame just like human MLH3 isoform 2.Consequently, without being bound by theory, it was anticipated that theSSOs targeting this exon to produce results like we found in hMLH3.

Due, in part, to the lack of reported mMlh3 isoform 2 homologues, otherexons were also targeted in order to have several viable candidate SSOsto put into the mouse. This provides a backup: 1) in case mMlh3 exon 6was not as easy to skip as hMLH3 exon 7 and 2) as mentioned in theoriginal grant, the literature indicate that rare adverse effects ofvivo-morpholinos may be mediated by sequence-specific interactions ofthe morpholinos that cause them to aggregate in the bloodstream (1).Consequently, the initial strategy was to target exons 5 and 7 as wellas exon 6 in the mouse MLH3 pre-mRNA. The graphic in FIG. 12 serves tovisualize that strategy. Optimally, each SSO would serve to exclude theexon it targets, however, in practice a range in efficacy has beenidentified in the case of human MLH3, and the same is expected in mice.Previously experiments demonstrate that using a pair of SSOs was moreeffective than a single SSO at the same total morpholino concentration(see FIG. 6 and FIG. 7).

Primary fibroblasts derived from the C57BL/6 mouse were originallyproposed to be used to test the SSOs. Part of the reasoning for using anisogenic line was to avoid the possibility that private point mutationsin a cell line would interfere with results. Unfortunately, the primaryfibroblasts expressed little mMlh3, making the determination of spliceswitching much more difficult than it had been in the HEK293 cells usedin human experiments. NIH3T3 cell line was used, as this cell line mightwork better. The primary and transformed mouse fibroblastic cells gavesimilar, if not presentable, results, allowing for decisions to be madeabout SSO efficacy and excluded the mMlh3 exon 7-targeted SSO mMlh3dr8from further consideration.

Without be bound by theory, the ability to study repeat expansion andhMLH3 isoforms in HEK293 cells was aided by the neuronal nature of theHEK293 line (2,3). Therefore, after working with fibroblasts, mouse celllines with a neuronal nature were sought for future experiments. A mouseneuroblastoma cell line called Neuro-2A (4) expressed sufficient mMlh3for us to complete the testing of the candidate SSOs. For example,experiments such as that shown in FIG. 13 were used to refine doses inorder to look at possible synergies between the SSOs. In general, theSSO treatments resulted in the discrete fragments predicted.

For instance, in lanes 4 and 5 in FIG. 13, there are 3 bandscorresponding to the 4 possible fragments indicated to the right of thegel image. The bands within each lane are fairly quantitative relativeto one another because they are in competition for the same primers, butwe did not control for loading between lanes. However, the consistentlylower yield of products with use of mMlh3ac4 was verified with real-timePCR quantification of mMlh3 message. Without being bound by theory, thisreproducible several fold reduction was related to nonsense-mediateddecay due to the frame-shift caused by loss of the 73 base long exon 5.In contrast, skipping exon 6 leaves the reading frame intact.

Although the morpholino mMlh3ac4 was more effective than any othersingle morpholino in reducing the amount of full-length mMlh3 mRNA, itwas not used in mice for several reasons. First, and foremost, thecombination of SSOs mMlh3ac5 and mMlh3dr7 produced a reliable switch tothe mouse equivalent of hMLH3 isoform 2. This pair has the dualadvantages of closely mimicking what will be accomplish in FRDApatients, and not causing degradation of the mMlh3 mRNA, so that theratio of the isoforms can more readily be detected. In contrast, use ofmMlh3ac4 complicated the assays because of the degradation of mMlh3 exon5-skipped mRNA. In mouse fibroblasts, the products became difficult todetect.

Morpholinos can Sustain mMlh3 Splice Switching in the Mouse.

The FRDA mouse model work is underway and results demonstrate that themouse versions of the SSOs penetrate tissue and change the mouse MLH3splicing pattern (FIG. 14). Ongoing studies in the mouse model willtrack and correlate the expansion of FRDA repeats over time to provideproof of therapeutic efficacy, which will enhance pharmaceuticalinterest. The results of a test for splice-switching activity in C57BL/6mice are shown in FIG. 14.

Injection of Higher Doses of In Vivo Morpholinos in Mice Demonstrate NoNoticable Adverse Reactions.

A rare, but serious side effect was the possibility that one of the InVivo-morpholinos would clump and cause a toxic clot in the animals. Inthe literature, such an adverse event is usually fatal within a fewminutes (1). Such a reaction is more likely at a dose above 12 μg/g. Sofar, no mice have exhibited such adverse events at even 50 μg/g.

The higher dose was more effective, switching a relatively greaterfraction of the mMlh3 to isoform 2. Coupled with the lack of adverseevents, future experiments will use the higher dose. Finally, becausecurrent formulations do not cross the blood brain barrier, the SSOs willbe injected directly into brain for future experiments.

Murine SSO Sequences Tested in this Example

Oligo Identifier name Nucleotide Sequence SEQ ID NO: 45 mMlh3ac4GAACCTGCGATTCACGGAGATAAGT SEQ ID NO: 46 mMlh3ac5TCCACCTACAAAATAATCCAGGATT SEQ ID NO: 47 mMlh3dr7AACTACAGACAGATACTTACCAGTA SEQ ID NO: 48 mMlh3dr8CATGTCCTCAGGCTACTGACCGTAA

Murine Mlh3

The Mus musculus mutL homolog 3 (Mlh3) gene comprises approximately36,126 bp contained within the genomic region (GRCm38/mm10) Assemblychr12:85,234,466-85,270,591. The chromosomal location of murine Mlh3 isAccession No: NC_000078.6.

The two major murine Mlh3 variants (mMlh3) comprise:

1) Mus musculus mutL homolog 3 (Mlh3), transcript variant 1, mRNA NCBIReference Sequence: NM_175337.2

2) Mus musculus mutL homolog 3 (Mlh3), transcript variant 2, mRNA NCBIReference Sequence: NM_001304475.1

The two major human Mlh3 variants comprise:

1) RefSeq: NM_001040108.1 Homo sapiens mutL homolog 3 (MLH3), transcriptvariant 1, mRNA.

2) RefSeq: NM_014381.2 Homo sapiens mutL homolog 3 (MLH3), transcriptvariant 2, mRNA.

REFERENCES CITED IN THIS EXAMPLE

-   1) Ferguson, D. P., Dangott, L. J. and Lightfoot, J. T. (2014)    Lessons learned from vivo-morpholinos: How to avoid vivo-morpholino    toxicity. BioTechniques, 56, 251-256.-   2) Graham, F. L., Smiley, J., Russell, W. C. and Nairn, R. (1977)    Characteristics of a human cell line transformed by DNA from human    adenovirus type 5. The Journal of general virology, 36, 59-74.-   3) Shaw, G., Morse, S., Ararat, M. and Graham, F. L. (2002)    Preferential transformation of human neuronal cells by human    adenoviruses and the origin of HEK 293 cells. The FASEB journal:    official publication of the Federation of American Societies for    Experimental Biology, 16, 869-871.-   4) Olmsted, J. B., Carlson, K., Klebe, R., Ruddle, F. and    Rosenbaum, J. (1970) Isolation of microtubule protein from cultured    mouse neuroblastoma cells. Proceedings of the National Academy of    Sciences of the United States of America, 65, 129-136.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific substances and procedures described herein. Such equivalentsare considered to be within the scope of this invention, and are coveredby the following claims.

What is claimed:
 1. An isolated nuclease-resistant oligonucleotidecomprising a nucleic acid sequence that hybridizes to a complementarytarget nucleic acid sequence of a gene or gene product to modulate theexpression of a component of a mismatch repair (MMR) complex, whereinthe component of the MMR complex comprises exon 7 of MLH3 of MutLgamma,further wherein the isolated nuclease resistant oligonucleotide does notimpact the total cellular ratios of MLH1 and its binding partners. 2.The nuclease-resistant oligonucleotide of claim 1, wherein MLH3comprises SEQ ID NO:
 1. 3. The nuclease-resistant oligonucleotide ofclaim 1, wherein the oligonucleotide directs skipping of exon 7 of MLH3.4. The nuclease-resistant oligonucleotide of claim 1, wherein theoligonucleotide hybridizes to the target complementary nucleic acidsequence comprising SEQ ID NO:
 2. 5. The nuclease-resistantoligonucleotide of claim 1, wherein the oligonucleotide is at least 80%identical to a nucleic acid sequence comprising SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ IDNO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25,SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO:
 28. 6. Thenuclease-resistant oligonucleotide of claim 1, wherein theoligonucleotide comprises one or more morpholino subunits, one or morelocked nucleic acid subunits, one or more 2-O-methyl moieties, or one ormore peptide moieties.
 7. A pharmaceutical composition comprising anuclease-resistant oligonucleotide 15 to 30 nucleotide bases in lengthtargeted to a complementary nucleic acid sequence of exon 7 a geneencoding a MLH3 subunit of MutLgamma, wherein the oligonucleotidehybridizes with and modulates the expression of the human MutL subunitby at least 20%, wherein the oligonucleotide comprises at least onemodification, and further wherein the nuclease resistant oligonucleotidedoes not impact the total cellular ratios of MLH1 and its bindingpartners.
 8. The pharmaceutical composition of claim 7, wherein themodification comprises a phosphorothioate backbone, a phosphorodiamidatemorpholino nucleotide, a charge-negative oligonucleotide, acharge-neutral oligonucleotide, a 2-aminoethylglycine functionalizednucleotide phosphorothioate backbone, a 5-methylcytosine nucleotide, a2′-O-methoxyethyl sugar moiety, a locked nucleic acid subunit, anethylene-bridged nucleic acid subunit, or a combination thereof.
 9. Anoligonucleotide complex for modulating the expression or activity of agene or gene product encoding a component of a mismatch repair (MMR)system, the complex comprising a first oligonucleotide and a secondoligonucleotide, wherein the first oligonucleotide comprises a sequencecomplementary to an acceptor region of exon 7 of a gene encoding a MLH3subunit, and wherein the first oligonucleotide comprises anuclease-resistant modification, and wherein the second oligonucleotidecomprises a sequence complementary to a donor region of exon 7 of a geneencoding a MLH3 subunit, and wherein the second oligonucleotidecomprises a nuclease-resistant modification.
 10. An oligonucleotidecomplex for modulating the expression or activity of a gene or geneproduct encoding a component of a mismatch repair (MMR) system, thecomplex comprising a first oligonucleotide and a second oligonucleotide,wherein the first oligonucleotide comprises a sequence complementary toan acceptor region of exon 7 of a gene encoding a MLH3 subunit, andwherein the first oligonucleotide comprises a nuclease-resistantmodification, and wherein the second oligonucleotide comprises asequence complementary to a donor region of exon 7 of a gene encoding aMLH3 subunit.
 11. An oligonucleotide complex for modulating theexpression or activity of a gene or gene product encoding a component ofa mismatch repair (MMR) system, the complex comprising a firstoligonucleotide and a second oligonucleotide, wherein the firstoligonucleotide comprises a sequence complementary to an acceptor regionof exon 7 of a gene encoding a MLH3 subunit, and wherein the secondoligonucleotide comprises a sequence complementary to a donor region ofexon 7 of a gene encoding a MLH3 subunit, and wherein the secondoligonucleotide comprises a nuclease-resistant modification.
 12. Theoligonucleotide complex of claim 9, 10, or 11, wherein thenuclease-resistant modification comprises one or more morpholinosubunits, one or more locked nucleic acid subunits, one or more2-O-methyl moieties, one or more peptide moieties, or a combinationthereof.
 13. The oligonucleotide complex of claim 9, 10, or 11, whereinthe first or second oligonucleotide comprises a nucleic acid sequencehaving at least 90% identity to SEQ ID NO: 3 or SEQ ID NO:
 4. 14. Thecomposition of claim 7, wherein the oligonucleotide decreases theexpression of the human MutL subunit by at least 20%.
 15. Thenuclease-resistant oligonucleotide of claim 1, wherein theoligonucleotide comprises a pharmaceutical composition administered to asubject in a pharmaceutically acceptable carrier.